Protein variants having modified immunogenicity

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application of PCT/DK01/00293 filed Apr. 30, 2001 and claims, under 35 U.S.C. 119, priority or the benefit of Danish application nos. PA 2000 00707 and PA 2001 00327 filed Apr. 28, 2000 and Feb. 28, 2001, respectively, and U.S. application Ser. Nos. 60/203,345 and 60/277,817 filed May 10, 2000 and Mar. 21, 2001, respectively, the contents of which are fully incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to a method of selecting a protein variant having modified immunogenicity as compared to the parent protein, to the protein variant and use thereof, as well as to a method for producing said protein variant.

BACKGROUND OF THE INVENTION

An increasing number of proteins, including enzymes, are being produced industrially, for use in various industries, housekeeping and medicine. Being proteins they are likely to stimulate an immunological response in man and animals, including an allergic response.

Depending on the application, individuals get sensitised to the respective allergens by inhalation, direct contact with skin and eyes, or injection. The general mechanism behind an allergic response is divided in a sensitisation phase and a symptomatic phase. The sensitisation phase involves a first exposure of an individual to an allergen. This event activates specific T- and B-lymphocytes, and leads to the production of allergen specific IgE antibodies (in the present context the antibodies are denoted as usual, i.e. immunoglobulin E is IgE etc.). These IgE antibodies eventually facilitate allergen capturing and presentation to T-lymphocytes at the onset of the symptomatic phase. This phase is initiated by a second exposure to the same or a resembling antigen. The specific IgE antibodies bind to the specific IgE receptors on mast cells and basophils, among others, and capture at the same time the allergen. The polyclonal nature of this process results in bridging and clustering of the IgE receptors, and subsequently in the activation of mast cells and basophils. This activation triggers the release of various chemical mediators involved in the early as well as late phase reactions of the symptomatic phase of allergy. Prevention of allergy in susceptible individuals is therefore a research area of great importance.

For certain forms of IgE-mediated allergies, a therapy exists, which comprises repeated administration of allergen preparations called ‘allergen vaccines’ (Int. Arch. Allergy Immunol., 1999, vol. 119, pp1-5). This leads to reduction of the allergic symptoms, possibly due to a redirection of the immune response away from the allergic (Th2) pathway and towards the immunoprotective (Th1) pathway (Int. Arch. Allergy Immunol., 1999, vol. 119, pp1-5).

Various attempts to reduce the immunogenicity of polypeptides and proteins have been conducted. It has been found that small changes in an epitope may affect the binding to an antibody. This may result in a reduced importance of such an epitope, maybe converting it from a high affinity to a low affinity epitope, or maybe even result in epitope loss, i.e. that the epitope cannot sufficiently bind an antibody to elicit an immunogenic response.

There is a need for methods to identify epitopes on proteins and alter these epitopes in order to modify the immunogenicity of proteins in a targeted manner. Such methods and kits for their execution can have at least four useful purposes:

1) reduce the allergenicity of a commercial protein using protein engineering.

2) reduce the potential of commercial proteins to cross-react with environmental allergens and hence cause allergic reactions in people sensitized to the environmental allergens (or vice versa).

3) improve the immunotherapeutic effect of allergen vaccines.

4) assist characterization of clinical allergies in order to select the appropriate treatment, including allergen vaccination.

In WO 99/53038 (Genencor Int.) as well as in prior references (Kammerer et al, Clin. Exp. Allergy, 1997, vol. 27, pp 1016-1026; Sakakibara et al, J. Vet. Med. Sci., 1998; vol. 60, pp. 599-605), methods are described, which identify linear T-cell epitopes among a library of known peptide sequences, each representing part of the primary sequence of the protein of interest. Further, several similar techniques for localization of B-cell epitopes are disclosed by Walshet et al, J. Immunol. Methods, vol.121, 1275-280, (1989), and by Schoofs et al. J. Immunol. vol.140, 611-616, (1987). All of these methods, however, only leads to identification of linear epitopes, not to identification of ‘structural’ or ‘discontinuous’ epitopes, which are found on the 3-dimensional surface of protein molecules and which comprise amino acids from several discrete sites of the primary sequence of the protein. For several allergens, it has been realized that the dominant epitopes are of such discontinuous nature (Collins et al., Clin. Exp. All. 1996, vol. 26, pp. 36-42).

Slootstra et al; Molecular Diversity, 2, pp. 156-164, 1996 disclose the screening of a semi-random library of synthetic peptides for their binding properties to three monoclonal antibodies by immobilizing the peptides on polyethylene pins and binding a dilution series of each antibody to the pins. This reference does not disclose any indication of how the antibody binding peptide sequences relate to any full protein antigens or allergens.

In WO 92/10755 a method for modifying proteins to obtain less immunogenic variants is described. Randomly constructed protein variants, revealing a reduced binding of antibodies to the parent enzyme as compared to the parent enzyme itself, are selected for the measurement in animal models in terms of allergenicity. Finally, it is assessed whether reduction in immunogenicity is due to true elimination of an epitope or a reduction in affinity for antibodies. This method targets the identification of amino acids that may be part of structural epitopes by using a complete protein for assessing antigen binding. The major drawbacks of this approach are the ‘trial and error’ character, which makes it a lengthy and expensive process, and the lack of general information on the epitope patterns. Without this information, the results obtained for one protein can not be applied on another protein.

WO 99/47680 (ALK-ABELLÓ) discloses the identification and modification of B-cell epitopes by protein engineering. However, the method is based on crystal structures of Fab-antigen complexes, and B-cell epitopes are defined as “a section of the surface of the antigen comprising 15-25 amino acid residues, which are within a distance from the atoms of the antibody enabling direct interaction” (p.3). This publication does not show how one selects which Fab fragment to use (e.g. to target the most dominant allergy epitopes) or how one selects the substitutions to be made. Further, their method cannot be used in the absence of such crystallographic data for antigen-antibody complexes, which are very cumbersome, sometimes impossible, to obtain—especially since one would need a separate crystal structure for each epitope to be changed.

Hence, it is of interest to establish a general and efficient method to identify structural epitopes on the 3-dimensional surface of commercial and environmental allergens.

SUMMARY OF THE INVENTION

The present invention relates to a method of selecting a protein variant having modified immunogenicity as compared to a parent protein, comprising the steps of:

a) obtaining antibody binding peptide sequences,

b) using the sequences to localise epitope sequences on the 3-dimensional structure of parent protein,

c) defining an epitope area including amino acids situated within 5 Å from the epitope amino acids constituting the epitope sequence,

d) changing one or more of the amino acids defining the epitope area of the parent protein by genetic engineering mutations of a DNA sequence encoding the parent protein,

e) introducing the mutated DNA sequence into a suitable host, culturing said host and expressing the protein variant, and

f) evaluating the immunogenicity of the protein variant using the parent protein as reference.

A second aspect of the present invention is a protein variant having modified immunogenicity as compared to its parent protein. The amino acid sequence of the protein variant differs from the amino acid sequence of the parent protein with respect to at least one epitope pattern of the parent protein, such that the immunogenicity of the protein variant is modified as compared with the immunogenicity of the parent protein.

A further aspect of the present invention is a composition comprising a protein variant as defined above, as well as the use of the composition for industrial application, such as the production of a formulation for personal care products (for example shampoo; soap; skin, hand and face lotions; skin, hand and face cremes; hair dyes; toothpaste), food (for example in the baking industry), detergents and for the production of pharmaceuticals, e.g. vaccines.

Yet another aspect is a DNA molecule encoding a protein variant as defined above.

Further aspects are a vector comprising a DNA molecule as described above as well a host cell comprising said DNA molecule.

Another aspect is a method of producing a protein variant having modified immunogenicity as compared to the parent protein as defined above.

Definitions

Prior to a discussion of the detailed embodiments of the invention, a definition of specific terms related to the main aspects of the invention is provided.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 1989”) DNA Cloning: A Practical Approach, Volumes I and II/D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984).

When applied to a protein, the term “isolated” indicates that the protein is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated protein is substantially free of other proteins, particularly other proteins of animal origin. It is preferred to provide the proteins in a highly purified form, i.e., greater than 95% pure, more preferably greater than 99% pure. When applied to a polynucleotide molecule, the term “isolated” indicates that the molecule is removed from its natural genetic milieu, and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, and may include naturally occurring 5′ and 3′ untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316: 774-78, 1985).

A “polynucleotide” is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.

A “nucleic acid molecule” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”) in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary or quaternary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.

A DNA “coding sequence” is a double-stranded DNA sequence, which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.

An “Expression vector” is a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and optionally one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences.

A “secretory signal sequence” is a DNA sequence that encodes a polypeptide (a “secretory peptide” that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.

The term “promoter” is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5′ non-coding regions of genes.

“Operably linked”, when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in the promoter and proceeds through the coding segment to the terminator.

A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.

“Isolated polypeptide” is a polypeptide which is essentially free of other non-[enzyme] polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE.

“Heterologous” DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. Preferably, the heterologous DNA includes a gene foreign to the cell.

A cell has been “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell. A cell has been “transformed” by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change. Preferably, the transforming DNA should be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.

A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. “Homologous recombination” refers to the insertion of a foreign DNA sequence of a vector in a chromosome. Preferably, the vector targets a specific chromosomal site for homologous recombination. For specific homologous recombination, the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.

Nucleic Acid Sequence

The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used. The nucleic acid sequence may be cloned from a strain producing the polypeptide, or from another related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleic acid sequence.

The term “isolated” nucleic acid sequence as used herein refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by agarose gel electorphoresis. For example, an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

Nucleic Acid Construct

As used herein the term “nucleic acid construct” is intended to indicate any nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA origin. The term “construct” is intended to indicate a nucleic acid segment which may be single- or double-stranded, and which may be based on a complete or partial naturally occurring nucleotide sequence encoding a polypeptide of interest. The construct may optionally contain other nucleic acid segments.

The DNA of interest may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., supra).

The nucleic acid construct may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801-805. According to the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.

Furthermore, the nucleic acid construct may be of mixed synthetic and genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire nucleic acid construct, in accordance with standard techniques.

The nucleic acid construct may also be prepared by polymerase chain reaction using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or Saiki et al., Science 239 (1988), 487-491.

The term nucleic acid construct may be synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention. The term “coding sequence” as defined herein is a sequence which is transcribed into mRNA and translated into a polypeptide of the present invention when placed under the control of the above mentioned control sequences. The boundaries of the coding sequence are generally determined by a translation start codon ATG at the 5′-terminus and a translation stop codon at the 3′-terminus. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

The term “control sequences” is defined herein to include all components which are necessary or advantageous for expression of the coding sequence of the nucleic acid sequence. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleic acid sequence which is recognized by a host cell for expression of the nucleic acid sequence. The promoter sequence contains transcription and translation control sequences which mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

The control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3′ terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.

The control sequence may also be a signal peptide coding region, which codes for an amino acid sequence linked to the amino terminus of the polypeptide which can direct the expressed polypeptide into the cell's secretory pathway of the host cell. The 5′ end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide.

Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding region which is foreign to that portion of the coding sequence which encodes the secreted polypeptide. A foreign signal peptide coding region may be required where the coding sequence does not normally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to obtain enhanced secretion relative to the natural signal peptide coding region normally associated with the coding sequence. The signal peptide coding region may be obtained from a glucoamylase or an amylase gene from an Aspergillus species, a lipase or proteinase gene from a Rhizomucor species, the gene for the alpha-factor from Saccharomyces cerevisiae, an amylase or a protease gene from a Bacillus species, or the calf preprochymosin gene. However, any signal peptide coding region capable of directing the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.

The control sequence may also be a propeptide coding region, which codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the Bacillus subtilis alkaline protease gene (aprE), the Bacillus subtilis neutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factor gene, or the Myceliophthora thermophilum laccase gene (WO 95/33836).

The nucleic acid constructs of the present invention may also comprise one or more nucleic acid sequences which encode one or more factors that are advantageous in the expression of the polypeptide, e.g., an activator (e.g., a trans-acting factor), a chaperone, and a processing protease. Any factor that is functional in the host cell of choice may be used in the present invention. The nucleic acids encoding one or more of these factors are not necessarily in tandem with the nucleic acid sequence encoding the polypeptide.

An activator is a protein which activates transcription of a nucleic acid sequence encoding a polypeptide (Kudla et al., 1990, EMBO Journal 9:1355-1364; Jarai and Buxton, 1994, Current Genetics 26:2238-244; Verdier, 1990, Yeast 6:271-297). The nucleic acid sequence encoding an activator may be obtained from the genes encoding Bacillus stearothermophilus NprA (nprA), Saccharomyces cerevisiae heme activator protein 1 (hap1), Saccharomyces cerevisiae galactose metabolizing protein 4 (gal4), and Aspergillus nidulans ammonia regulation protein (areA). For further examples, see Verdier, 1990, supra and MacKenzie et al., 1993, Journal of General Microbiology 139:2295-2307.

A chaperone is a protein which assists another polypeptide in folding properly (Harti et al., 1994, TIBS 19:20-25; Bergeron et al., 1994, TIBS 19:124-128; Demolder et al., 1994, Journal of Biotechnology 32:179-189; Craig, 1993, Science 260:1902-1903; Gething and Sambrook, 1992, Nature 355:33-45; Puig and Gilbert, 1994, Journal of Biological Chemistry 269:7764-7771; Wang and Tsou, 1993, The FASEB Journal 7:1515-11157; Robinson et al., 1994, Bio/Technology 1:381-384). The nucleic acid sequence encoding a chaperone may be obtained from the genes encoding Bacillus subtilis GroE proteins, Aspergillus oryzae protein disulphide isomerase, Saccharomyces cerevisiae calnexin, Saccharomyces cerevisiae BiP/GRP78, and Saccharomyces cerevisiae Hsp7O. For further examples, see Gething and Sambrook, 1992, supra, and Hartl et al., 1994, supra.

A processing protease is a protease that cleaves a propeptide to generate a mature biochemically active polypeptide (Enderlin and Ogrydziak, 1994, Yeast 10:67-79; Fuller et al., 1989, Proceedings of the National Academy of Sciences USA 86:1434-1438; Julius et al., 1984, Cell 37:1075-1089; Julius et al., 1983, Cell 32:839-852). The nucleic acid sequence encoding a processing protease may be obtained from the genes encoding Aspergillus niger Kex2, Saccharomyces cerevisiae dipeptidylaminopeptidase, Saccharomyces cerevisiae Kex2, and Yarrowia lipolytica dibasic processing endoprotease (xpr6).

It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems would include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and the Aspergillus olyzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those which allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene which is amplified in the presence of methotrexate, and the metallothionein genes which are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the polypeptide would be placed in tandem with the regulatory sequence.

Promoters

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E. coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus subtilis alkaline protease gene, the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75:3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80:21-25), or the Bacillus pumilus xylosidase gene, or by the phage Lambda PR or PL promoters or the E. coli lac, trp or tac promoters. Further promoters are described in “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242:74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (as described in U.S. Pat. No. 4,288,627, which is incorporated herein by reference), and hybrids thereof. Particularly preferred promoters for use in filamentous fungal host cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and glaA promoters. Further suitable promoters for use in filamentous fungus host cells are the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093-2099) or the tpiA promoter.

Examples of suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419-434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPI1 (U.S. Pat. No. 4,599,311) or ADH2-4c (Russell et al., Nature 304 1983), 652-654) promoters.

Further useful promoters are obtained from the Saccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae galactokinase gene (GAL1), the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488. In a mammalian host cell, useful promoters include viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).

Examples of suitable promoters for directing the transcription of the DNA encoding the polypeptide of the invention in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814) or the adenovirus 2 major late promoter.

An example of a suitable promoter for use in insect cells is the polyhedrin promoter (U.S. Pat. No. 4,745,051; Vasuvedan et al., FEBS Lett. 311, (1992) 7-11), the P10 promoter (J. M. VIak et al., J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica polyhedrosis virus basic protein promoter (EP 397 485), the baculovirus immediate early gene 1 promoter (U.S. Pat. No. 5,155,037; U.S. Pat No. 5,162,222), or the baculovirus 39K delayed-early gene promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No. 5,162,222).

Terminators

Preferred terminators for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. for fungal hosts) the TPI1 (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) terminators.

Preferred terminators for yeast host cells are obtained from the genes encoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), or Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

Polyadenylation Signals

Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, and Aspergillus niger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15:5983-5990.

Polyadenylation sequences are well known in the art for mammalian host cells such as SV40 or the adenovirus 5 Elb region.

Signal Sequences

An effective signal peptide coding region for bacterial host cells is the signal peptide coding region obtained from the maltogenic amylase gene from Bacillus NCIB 11837, the Bacillus stearothermophilus alpha-amylase gene, the Bacillus licheniformis subtilisin gene, the Bacillus licheniformis beta-lactamase gene, the Bacillus stearothermophilus neutral proteases genes (nprT, nprS, nprM), and the Bacillus subtilis PrsA gene. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

An effective signal peptide coding region for filamentous fungal host cells is the signal peptide coding region obtained from Aspergillus oryzae TAKA amylase gene, Aspergillus niger neutral amylase gene, the Rhizomucor miehei aspartic proteinase gene, the Humicola lanuginosa cellulase or lipase gene, or the Rhizomucor miehei lipase or protease gene, Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease. The signal peptide is preferably derived from a gene encoding A. oryzae TAKA amylase, A. niger neutral alpha-amylase, A. niger acid-stable amylase, or A. niger glucoamylase.

Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are described by Romanos et al., 1992, supra.

For secretion from yeast cells, the secretory signal sequence may encode any signal peptide which ensures efficient direction of the expressed polypeptide into the secretory pathway of the cell. The signal peptide may be naturally occurring signal peptide, or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides have been found to be the a-factor signal peptide (cf. U.S. Pat. No. 4,870,008), the signal peptide of mouse salivary amylase (cf. O Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modified carboxypeptidase signal peptide (cf. L. A. Valls et al., Cell 48, 1987, pp. 887-897), the yeast BAR1 signal peptide (cf. WO 87/02670), or the yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani et al., east 6, 1990, pp. 127-137).

For efficient secretion in yeast, a sequence encoding a leader peptide may also be inserted downstream of the signal sequence and uptream of the DNA sequence encoding the polypeptide. The function of the leader peptide is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e. exportation of the polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast cell). The leader peptide may be the yeast a-factor leader (the use of which is described in e.g. U.S. Pat. No. 4,546,082, EP 16 201, EP 123 294, EP 123 544 and EP 163 529). Alternatively, the leader peptide may be a synthetic leader peptide, which is to say a leader peptide not found in nature. Synthetic leader peptides may, for instance, be constructed as described in WO 89/02463 or WO 92/11378.

For use in insect cells, the signal peptide may conveniently be derived from an insect gene (cf. WO 90/05783), such as the lepidopteran Manduca sexta adipokinetic hormone precursor signal peptide (cf. U.S. Pat. No. 5,023,328).

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a nucleic acid sequence of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites. Alternatively, the nucleic acid sequence of the present invention may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression, and possibly secretion.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. The vector system may be a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.

The vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, tetracycline, neomycin, hygromycin or methotrexate resistance. A frequently used mammalian marker is the dihydrofolate reductase gene (DHFR). Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), and glufosinate resistance markers, as well as equivalents from other species. Preferred for use in an Aspergillus cell are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus. Furthermore, selection may be accomplished by co-transformation, e.g., as described in WO 91/17243, where the selectable marker is on a separate vector.

The vectors of the present invention preferably contain an element(s) that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome of the cell.

The vectors of the present invention may be integrated into the host cell genome when introduced into a host cell. For integration, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination. These nucleic acid sequences may be any sequence that is homologous with a target sequence in the genome of the host cell, and, furthermore, may be non-encoding or encoding sequences.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, pACYC184, pUB110, pE194, pTA1060, and pAMβ1. Examples of origin of replications for use in a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARS1. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433).

More than one copy of a nucleic acid sequence encoding a polypeptide of the present invention may be inserted into the host cell to amplify expression of the nucleic acid sequence. Stable amplification of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome using methods well known in the art and selecting for transformants.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

Host Cells

The present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides. The term “host cell” encompasses any progeny of a parent cell which is not identical to the parent cell due to mutations that occur during replication.

The cell is preferably transformed with a vector comprising a nucleic acid sequence of the invention followed by integration of the vector into the host chromosome. “Transformation” means introducing a vector comprising a nucleic acid sequence of the present invention into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. Integration is generally considered to be an advantage as the nucleic acid sequence is more likely to be stably maintained in the cell. Integration of the vector into the host chromosome may occur by homologous or non-homologous recombination as described above.

The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E. coli and Pseudomonas sp. In a preferred embodiment, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. The transformation of a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168:111-115), by using competent cells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnar and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169:5771-5278).

The host cell may be a eukaryote, such as a mammalian cell, an insect cell, a plant cell or a fungal cell.

Useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection.

Examples of suitable mammalian cell lines are the COS (ATCC CRL 1650 and 1651), BHK (ATCC CRL 1632, 10314 and 1573, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCC CCL 61) cell lines. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. AppI. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., N.Y., 1987, Hawley-Nelson et al., Focus 15 (1993), 73; Ciccarone et al., Focus 15 (1993), 80; Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J.1 (1982), 841-845.

In a preferred embodiment, the host cell is a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra). Representative groups of Ascomycota include, e.g., Neurospora, Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotium (=Aspergillus), and the true yeasts listed above. Examples of Basidiomycota include mushrooms, rusts, and smuts. Representative groups of Chytridiomycota include, e.g., Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi. Representative groups of Oomycota include, e.g., Saprolegniomycetous aquatic fungi (water molds) such as Achlya. Examples of mitosporic fungi include Aspergillus, Penicillium, Candida, and Alternaria. Representative groups of Zygomycota include, e.g., Rhizopus and Mucor.

In a preferred embodiment, the fungal host cell is a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera Pichia, Kluyveromyces and Saccharomyces). The basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeast belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e.g., genera Sorobolomyces and Bullera) and Cryptococcaceae (e.g., genus Candida). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980. The biology of yeast and manipulation of yeast genetics are well known in the art (see, e.g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B. J., and Stopani, A. O. M., editors, 2nd edition, 1987; The Yeasts, Rose, A. H., and Harrison, J. S., editors, 2nd edition, 1987; and The Molecular Biology of the Yeast Saccharomyces, Strathern et al., editors, 1981).

The yeast host cell may be selected from a cell of a species of Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, or Yarrowia. In a preferred embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. Other useful yeast host cells are a Kluyveromyces lactis, Kluyveromyces fragilis, Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica, Schizosaccharomyces pombe, Ustilgo maylis, Candida maltose, Pichia guillermondii and Pichia methanolio cell (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; U.S. Pat. No. 4,882,279 and U.S. Pat. No. 4,879,231).

In a preferred embodiment, the fungal host cell is a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are characterized by a vegetative mycelium composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative. In a more preferred embodiment, the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, and Trichoderma or a teleomorph or synonym thereof. In an even more preferred embodiment, the filamentous fungal host cell is an Aspergillus cell. In another even more preferred embodiment, the filamentous fungal host cell is an Acremonium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Fusarium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Humicola cell. In another even more preferred embodiment, the filamentous fungal host cell is a Mucor cell. In another even more preferred embodiment, the filamentous fungal host cell is a Myceliophthora cell. In another even more preferred embodiment, the filamentous fungal host cell is a Neurospora cell. In another even more preferred embodiment, the filamentous fungal host cell is a Penicillium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Thielavia cell. In another even more preferred embodiment, the filamentous fungal host cell is a Tolypocladium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Trichoderma cell. In a most preferred embodiment, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger, Aspergillus nidulans or Aspergillus oryzae cell. In another most preferred embodiment, the filamentous fungal host cell is a Fusarium cell of the section Discolor (also known as the section Fusarium). For example, the filamentous fungal parent cell may be a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, or Fusarium trichothecioides cell. In another prefered embodiment, the filamentous fungal parent cell is a Fusarium strain of the section Elegans, e.g., Fusarium oxysporum. In another most preferred embodiment, the filamentous fungal host cell is a Humicola insolens or Humicola lanuginosa cell. In another most preferred embodiment, the filamentous fungal host cell is a Mucor miehei cell. In another most preferred embodiment, the filamentous fungal host cell is a Myceliophthora thermophilum cell. In another most preferred embodiment, the filamentous fungal host cell is a Neurospora crassa cell. In another most preferred embodiment, the filamentous fungal host cell is a Penicillium purpurogenum cell. In another most preferred embodiment, the filamentous fungal host cell is a Thielavia terrestris cell or an Acremonium chrysogenum cell. In another most preferred embodiment, the Trichoderma cell is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride cell. The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 230 023.

Transformation

Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 25 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81:1470-1474. A suitable method of transforming Fusarium species is described by Malardier et al., 1989, Gene 78:147-156 or in copending U.S. Ser. No. 08/269,449. Examples of other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., in particular strains of A. oryzae, A. nidulans or A. niger. The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 230 023. The transformation of F. oxysporum may, for instance, be carried out as described by Malardier et al., 1989, Gene 78: 147-156.

Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75:1920. Mammalian cells may be transformed by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52:546).

Transformation of insect cells and production of heterologous polypeptides therein may be performed as described in U.S. Pat. No. 4,745,051; U.S. Pat. No. 4,775,624; U.S. Pat. No. 4,879,236; U.S. Pat. No. 5,155,037; U.S. Pat. No. 5,162,222; EP 397,485) all of which are incorporated herein by reference. The insect cell line used as the host may suitably be a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. U.S. Pat. No. 5,077,214). Culture conditions may suitably be as described in, for instance, WO 89/01029 or WO 89/01028, or any of the aforementioned references.

Methods of Production

The transformed or transfected host cells described above are cultured in a suitable nutrient medium under conditions permitting the production of the desired molecules, after which these are recovered from the cells, or the culture broth.

The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The media are prepared using procedures known in the art (see, e.g., references for bacteria and yeast; Bennett, J. W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).

If the molecules are secreted into the nutrient medium, they can be recovered directly from the medium. If they are not secreted, they can be recovered from cell lysates. The molecules are recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of molecule in question.

The molecules of interest may be detected using methods known in the art that are specific for the molecules. These detection methods may include use of specific antibodies, formation of a product, or disappearance of a substrate. For example, an enzyme assay may be used to determine the activity of the molecule. Procedures for determining various kinds of activity are known in the art.

The molecules of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J -C Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

The term “immunological response”, used in connection with the present invention, is the response of an organism to a compound, which involves the immune system according to any of the four standard reactions (Type I, II, III and IV according to Coombs & Gell).

Correspondingly, the “immunogenicity” of a compound used in connection with the present invention refers to the ability of this compound to induce an ‘immunological response’ in animals including man.

The term “allergic response”, used in connection with the present invention, is the response of an organism to a compound, which involves IgE mediated responses (Type I reaction according to Coombs & Gell). It is to be understood that sensibilization (i.e. development of compound-specific IgE antibodies) upon exposure to the compound is included in the definition of “allergic response”.

Correspondingly, the “allergenicity” of a compound used in connection with the present invention refers to the ability of this compound to induce an ‘allergic response’ in animals including man.

The term “parent protein” refer to the polypeptide to be modified by creating a library of diversified mutants. The “parent protein” may be a naturally occurring (or wild-type) polypeptide or it may be a variant thereof prepared by any suitable means. For instance, the “parent protein” may be a variant of a naturally occurring polypeptide which has been modified by substitution, deletion or truncation of one or more amino acid residues or by addition or insertion of one or more amino acid residues to the amino acid sequence of a naturally-occurring polypeptide.

The term “enzyme variants” or “protein variants” refer to a polypeptide of the invention comprising one or more substitutions of the specified amino acid residues. The total number of such substitutions is typically not more than 10, e.g. one, two, three, four, five or six of said substitutions. In addition, the enzyme variant or protein variant of the invention may optionally include other modifications of the parent enzyme, typically not more than 10, e.g. not more than 5 such modifications. The variant generally has a homology with the parent enzyme of at least 80%, e.g. at least 85%, typically at least 90% or at least 95%.

The term “randomized library” of protein variants refers to a library with at least partially randomized composition of the members, e.g. protein variants.

An “epitope” is a set of amino acids on a protein that are involved in an immunological response, such as antibody binding or T-cell activation. One particularly useful method of identifying epitopes involved in antibody binding is to screen a library of peptide-phage membrane protein fusions and selecting those that bind to relevant antigen-specific antibodies, sequencing the randomized part of the fusion gene, aligning the sequences involved in binding, defining consensus sequences based on these alignments, and mapping these consensus sequences on the surface or the sequence and/or structure of the antigen, to identify epitopes involved in antibody binding.

By the term “epitope pattern” is meant such a consensus sequence of antibody binding peptides. An example is the epitope pattern A R R<R. The sign “<” in this notation indicates that the aligned antibody binding peptides included a non-consensus amino acid between the second and the third arginine.

An “epitope area” is defined as the amino acids situated close to the epitope sequence amino acids. Preferably, the amino acids of an epitope area are located <5 Å from the epitope sequence. Hence, an epitope area also includes the corresponding epitope sequence itself. Modifications of amino acids of the ‘epitope area’ can possibly affect the immunogenic function of the corresponding epitope.

By the term “epitope sequence” is meant the amino acid residues of a parent protein, which have been identified to belong to an epitope by the methods of the present invention (an example of an epitope sequence is E271 Q12 18 in Savinase).

The term ‘antibody binding peptide’ denotes a peptide that bind with sufficiently high affinity to antibodies. Identification of ‘antibody binding peptides’ and their sequences constitute the first step of the method of this invention.

“Anchor amino acids” are the individual amino acids of an epitope pattern.

“Hot spot amino acids” are amino acids of parent protein, which are particularly likely to result in modified immunogenecity if they are mutated. Amino acids, which appear in three or more epitope sequences or which correspond to anchor amino acids are hot spot amino acids.

“Environmental allergens” are protein allergens that are present naturally. They include pollen, dust mite allergens, pet allergens, food allergens, venoms, etc.

“Commercial allergens” are protein allergens that are being brought to the market commercially. They include enzymes, pharmaceutical proteins, antimicrobial peptides, as well as allergens of transgenic plants.

The “donor protein” is the protein that was used to raise antibodies used to identify antibody binding sequences, hence the donor protein provides the information that leads to the epitope patterns.

The “acceptor protein” is the protein, whose structure is used to fit the identified epitope patterns and/or to fit the antibody binding sequences. Hence the acceptor protein is also the parent protein.

An “autoepitope” is one that has been identified using antibodies raised against the parent protein, i.e. the acceptor and the donor proteins are identical.

A “heteroepitope” is one that has been identified with distinct donor and acceptor proteins.

The term “functionality” of protein variants refers to e.g. enzymatic activity; binding to a ligand or receptor; stimulation of a cellular response (e.g. ³H-thymidine incorporation as response to a mitogenic factor); or anti-microbial activity.

By the term “specific polyclonal antibodies” is meant polyclonal antibodies isolated according to their specificity for a certain antigen, e.g. the protein backbone.

By the term “monospecific antibodies” is meant polyclonal antibodies isolated according to their specificity for a certain epitope. Such monospecific antibodies will bind to the same epitope, but with different affinity, as they are produced by a number of antibody producing cells recognizing overlapping but not necessarily identical epitopes.

The term “randomized library” of protein variants refers to a library with at least partially randomized composition of the members, e.g. protein variants.

‘Spiked mutagenesis’ is a form of site-directed mutagenesis, in which the primers used have been synthesized using mixtures of oligonucleotides at one or more positions.

By the term “a protein variant having modified immunogenicity as compared to the parent protein” is meant a protein variant which differs from the parent protein in one or more amino acids whereby the immunogenicity of the variant is modified. The modification of immunogenicity may be confirmed by testing the ability of the protein variant to elicit an IgE/lgG response.

In the present context the term “protein” is intended to cover oligopeptides, polypeptides as well as proteins as such.

DETILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of selecting a protein variant having modified immunogenicity as compared to a parent protein, comprising the steps of:

a) obtaining antibody binding peptide sequences,

b) using the sequences to localise epitope sequences on the 3-dimensional structure of parent protein,

c) defining an epitope area including amino acids situated within 5 Å from the epitope amino acids constituting the epitope sequence,

d) changing one or more of the amino acids defining the epitope area of the parent protein by genetic engineering mutations of a DNA sequence encoding the parent protein,

e) introducing the mutated DNA sequence into a suitable host, culturing said host and expressing the protein variant, and

f) evaluating the immunogenicity of the protein variant using the parent protein as reference.

A) How to Find Antibody Binding Peptide Sequences and Epitope Patterns

A first step of the method is to identify peptide sequences, which bind specifically to antibodies.

Antibody binding peptide sequences can be found by testing a set of known peptide sequences for binding to antibodies raised against the donor protein. These sequences are typically selected, such that each represents a segment of the donor protein sequence (Mol. Immunol., 1992, vol. 29, pp. 1383-1389; Am. J. Resp. Cell. Mol. Biol. 2000, vol. 22, pp. 344-351). Also, randomized synthetic peptide libraries can be used to find antibody binding sequences (Slootstra et al; Molecular Diversity, 1996, vol. 2, pp. 156-164).

In a preferred method, the identification of antibody binding sequences may be achieved by screening of a display package library, preferably a phage display library. The principle behind phage display is that a heterologous DNA sequence can be inserted in the gene coding for a coat protein of the phage (WO 92/15679). The phage will make and display the hybrid protein on its surface where it can interact with specific target agents. Such target agent may be antigen-specific antibodies. It is therefore possible to select specific phages that display antibody-binding peptide sequences. The displayed peptides can be of predetermined lengths, for example 9 amino acids long, with randomized sequences, resulting in a random peptide display package library. Thus, by screening for antibody binding, one can isolate the peptide sequences that have sufficiently high affinity for the particular antibody used. The peptides of the hybrid proteins of the specific phages which bind protein-specific antibodies characterize epitopes that are recognized by the immune system.

The antibodies used for reacting with the display package are preferably lgE antibodies to ensure that the epitopes identified are IgE epitopes, i.e. epitopes inducing and binding IgE. In a preferred embodiment the antibodies are polyclonal antibodies, optionally monospecific antibodies.

For the purpose of the present invention polyclonal antibodies are preferred in order to obtain a broader knowledge about the epitopes of a protein.

It is of great importance that the amino acid sequence of the peptides presented by the display packages is long enough to represent a significant part of the epitope to be identified. In a preferred embodiment of the invention the peptides of the peptide display package library are oligopeptides having from 5 to 25 amino acids, preferably at least 8 amino acids, such as 9 amino acids. For a given length of peptide sequences (n), the theoretical number of different possible sequences can be calculated as 20ⁿ. The diversity of the package library used must be large enough to provide a suitable representation of the theoretical number of different sequences. In a phage-display library, each phage has one specific sequence of a determined length. Hence an average phage display library can express 10⁸−10¹² different random sequences, and is therefore well-suited to represent the theoretical number of different sequences.

The antibody binding peptide sequences can be further analysed by consensus alignment e.g. by the methods described by Feng and Doolittle, Meth. Enzymol., 1996, vol. 266, pp. 368-382; Feng and Doolittle, J. Mol. Evol., 1987, vol. 25, pp. 351-360; and Taylor,. Meth. Enzymol., 1996, vol. 266, pp. 343-367.

This leads to identification of epitope patterns, which can assist the comparison of the linear information obtained from the antibody binding peptide sequences to the 3-dimensional structure of the acceptor protein in order to identify epitope sequences at the surface of the acceptor protein.

B) How to Identify Epitope Sequences and Epitope Areas

Given a number of antibody binding peptide sequences and possibly the corresponding epitope patterns, one need the 3-dimensional structure coordinates of an acceptor protein to find the epitope sequences on its surface.

These coordinates can be found in databases (NCBI: http://www.ncbi.nlm.nih.gov/), determined experimentally using conventional methods (Ducruix and Giegé: Crystallization of Nucleic Acids and Proteins, IRL PRess, Oxford, 1992, ISBN 0-19-963245-6), or they can be deduced from the coordinates of a homologous protein. Typical actions required for the construction of a model structure are: alignment of homologous sequences for which 3-dimensional structures exist, definition of Structurally Conserved Regions (SCRs), assignment of coordinates to SCRs, search for structural fragments/loops in structure databases to replace Variable Regions, assignment of coordinates to these regions, and structural refinement by energy minimization. Regions containing large inserts (>3 residues) relative to the known 3-dimensional structures are known to be quite difficult to model, and structural predictions must be considered with care.

Using the coordinates and the several methods of mapping the linear information on the 3-dimensional surface are possible, as described in the examples below.

One can match each amino acid residue of the antibody binding peptide to an identical or homologous amino acid on the 3-D surface of the acceptor protein, such that amino acids that are adjacent in the primary sequence are close on the surface of the acceptor protein, with close being <5 Å, preferably <3 Å between any two atoms of the two amino acids.

Alternatively, one can define a geometric body (e.g. an ellipsoid, a sphere, or a box) of a size that matches a possible binding interface between antibody and antigen and look for a positioning of this body where it will contain most of or all the anchor amino acids.

Also, one can use the epitope patterns to facilitate identification of epitope sequences. This can be done, by first matching the anchor amino acids on the 3-D structure and subsequently looking for other elements of the antibody binding peptide sequences, which provide additional matches. If there are many residues to be matched, it is only necessary that a suitable number can be found on the 3-D structure. For example if an epitope pattern comprises 4, 5, 6, or 7 amino acids, it is only necessary that 3 matches surface elements of the acceptor protein.

In all cases, it is desirable that amino acids of the epitope sequence are surface exposed (as described below in Examples).

It is known, that amino acids that surround binding sequences can affect binding of a ligand without participating actively in the binding process. Based on this knowledge, areas covered by amino acids with potential steric effects on the epitope-antibody interaction, were defined around the identified epitope sequences. These areas are called ‘epitope areas’. Practically, all amino acids situated within 5 Å from the amino acids defining the epitope sequence were included. Preferably, the epitope area equals the epitope sequence. The accessibility criterium was not used as hidden amino acids of an epitope area also can have an effect on the adjacent amino acids of the epitope sequence.

C) How to Use the Epitope Information

There are at least four ways to utilize the information about epitope sequences, which has been derived by the methods of this invention:

1) reduce the allergenicity of a commercial protein using protein engineering.

2) reduce the potential of commercial proteins to cross-react with environmental allergens and hence cause allergic reactions in people sensitized to the environmental allergens (or vice versa).

3) improve the immunotherapeutic effect of allergen vaccines.

4) assist characterization of clinical allergies in order to select the appropriate allergen vaccine.

Protein Engineering to Reduce the Allergenicity, Cross-Reactivity and/or Immunotherapeutic Effect of Proteins

The methods described thus far have led to identification of epitope areas on an acceptor protein, each containing epitope sequences. These subsets of amino acids, are preferred for introducing mutations that are meant to modify the immunogenecity of the acceptor protein. An even more preferred subset of amino acids to target by mutagenesis are ‘hot spot amino acids’, which appear in several different epitope sequences, or which corresponds to anchor amino acids of the epitope patterns.

Thus, genetic engineering mutations should be designed in the epitope areas, preferably in epitope sequences, and more preferably in the ‘hot spot amino acids’.

Substitution, Deletion, Insertion

When the epitope area(s) have been identified, a protein variant exhibiting a modified immunogenicity may be produced by changing the identified epitope area of the parent protein by genetic engineering mutation of a DNA sequence encoding the parent protein.

The epitope identified may be changed by substituting at least one amino acid of the epitope area. In a preferred embodiment at least one anchor amino acid or hot spot amino acid is changed. The change will often be substituting to an amino acid of different size, hydrophilicity, and/or polarity, such as a small amino acid versus a large amino acid, a hydrophilic amino acid versus a hydrophobic amino acid, a polar amino acid versus a non-polar amino acid and a basic versus an acidic amino acid.

Other changes may be the addition/insertion or deletion of at least one amino acid of the epitope sequence, preferably deleting an anchor amino acid or a hot spot amino acid. Furthermore, an epitope pattern may be changed by substituting some amino acids, and deleting/adding other.

In the claims a position to be changed by substitution, insertion, deletion will be indicated by: “Position xx to aaa, bbb, ccc, insertion, deletion”, meaning that position xx can be substituted by the amino acid aaa, bbb, ccc or that any amino acid can be inserted after position xx or that position xx can be deleted, e.g. “Position 27 to A, D, E, insertion, deletion” means that in position 27 the amino acid can be substituted by A, D or E, or that any amino acid can be inserted after position 27, or that the amino acid in position 27 can be deleted.

When one uses protein engineering to eliminate epitopes, it is indeed possible that new epitopes are created, or existing epitopes are duplicated. To reduce this risk, one can map the planned mutations at a given position on the 3-dimensional structure of the protein of interest, and control the emerging amino acid constellation against a database of known epitope patterns, to rule out those possible replacement amino acids, which are predicted to result in creation or duplication of epitopes. Thus, risk mutations can be identified and eliminated by this procedure, thereby reducing the risk of making mutations that lead to increased rather than decreased allergenicity.

Introduction of Residues for Chemical Derivatization in Epitope Areas

In yet another embodiment, one can design the mutation, such that amino acids suitable for chemical modification are substituted for existing ones in the epitope areas. The protein variant can then be conjugated to activated polymers. Which amino acids to substitute and/or insert, depends in principle on the coupling chemistry to be applied. The chemistry for preparation of covalent bioconjugates can be found in “Bioconjugate Techniques”, Hermanson, G. T. (1996), Academic Press Inc., which is hereby incorporated as reference (see below). It is preferred to make conservative substitutions in the polypeptide when the polypeptide has to be conjugated, as conservative substitutions secure that the impact of the substitution on the polypeptide structure is limited. In the case of providing additional amino groups this may be done by substitution of arginine to lysine, both residues being positively charged, but only the lysine having a free amino group suitable as an attachment groups. In the case of providing additional carboxylic acid groups the conservative substitution may for instance be an asparagine to aspartic acid or glutamine to glutamic acid substitution. These residues resemble each other in size and shape, except from the carboxylic groups being present on the acidic residues. In the case of providing SH-groups the conservative substitution may be done by changing threonine or serine to cysteine.

Chemical Conjugation

For chemical conjugation, the protein variant needs to be incubate with an active or activated polymer and subsequently separated from the unreacted polymer. This can be done in solution followed by purification or it can conveniently be done using the immobilized protein variants, which can easily be exposed to different reaction environments and washes.

In the case were polymeric molecules are to be conjugated with the polypeptide in question and the polymeric molecules are not active they must be activated by the use of a suitable technique. It is also contemplated according to the invention to couple the polymeric molecules to the polypeptide through a linker. Suitable linkers are well-known to the skilled person. Methods and chemistry for activation of polymeric molecules as well as for conjugation of polypeptides are intensively described in the literature. Commonly used methods for activation of insoluble polymers include activation of functional groups with cyanogen bromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine etc. (see R. F. Taylor, (1991), “Protein immobilisation. Fundamental and applications”, Marcel Dekker, N.Y.; S. S. Wong, (1992), “Chemistry of Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T. Hermanson et al., (1993), “Immobilized Affinity Ligand Techniques”, Academic Press, N.Y.). Some of the methods concern activation of insoluble polymers but are also applicable to activation of soluble polymers e.g. periodate, trichlorotriazine, sulfonylhalides, divinylsulfone, carbodiimide etc. The functional groups being amino, hydroxyl, thiol, carboxyl, aldehyde or sulfydryl on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemistry which normally consist of i) activation of polymer, ii) conjugation, and iii) blocking of residual active groups.

In the following a number of suitable polymer activation methods will be described shortly. However, it is to be understood that also other methods may be used.

Coupling polymeric molecules to the free acid groups of polypeptides may be performed with the aid of diimide and for example amino-PEG or hydrazino-PEG (Pollak et al., (1976), J. Am. Chem. Soc., 98, 289-291) or diazoacetate/amide (Wong et al., (1992), “Chemistry of Protein Conjugation and Crosslinking”, CRC Press).

Coupling polymeric molecules to hydroxy groups is generally very difficult as it must be performed in water. Usually hydrolysis predominates over reaction with hydroxyl groups.

Coupling polymeric molecules to free sulfhydryl groups can be achieved with special groups like maleimido or the ortho-pyridyl disulfide. Also vinylsulfone (U.S. Pat. No. 5,414,135, (1995), Snow et al.) has a preference for sulfhydryl groups but is not as selective as the other mentioned.

Accessible arginine residues in the polypeptide chain may be targeted by groups comprising two vicinal carbonyl groups.

Techniques involving coupling of electrophilically activated PEGs to the amino groups of Lysines may also be useful. Many of the usual leaving groups for alcohols give rise to an amine linkage. For instance, alkyl sulfonates, such as tresylates (Nilsson et al., (1984), Methods in Enzymology vol. 104, Jacoby, W. B., Ed., Academic Press: Orlando, p. 56-66; Nilsson et al., (1987), Methods in Enzymology vol. 135; Mosbach, K., Ed.; Academic Press: Orlando, pp. 65-79; Scouten et al., (1987), Methods in Enzymology vol. 135, Mosbach, K., Ed., Academic Press: Orlando, 1987; pp 79-84; Crossland et al., (1971), J. Amr. Chem. Soc. 1971, 93, pp. 4217-4219), mesylates (Harris, (1985), supra; Harris et al., (1984), J. Polym. Sci. Polym. Chem. Ed. 22, pp 341-352), aryl sulfonates like tosylates, and para-nitrobenzene sulfonates can be used.

Organic sulfonyl chlorides, e.g. Tresyl chloride, effectively converts hydroxy groups in a number of polymers, e.g. PEG, into good leaving groups (sulfonates) that, when reacted with nucleophiles like amino groups in polypeptides allow stable linkages to be formed between polymer and polypeptide. In addition to high conjugation yields, the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity), and satisfy the non-destructive requirements to the polypeptide.

Tosylate is more reactive than the mesylate but also less stable decomposing into PEG, dioxane, and sulfonic acid (Zalipsky, (1995), Bioconjugate Chem., 6, 150-165). Epoxides may also been used for creating amine bonds but are much less reactive than the abovementioned groups.

Converting PEG into a chloroformate with phosgene gives rise to carbamate linkages to Lysines. Essentially the same reaction can be carried out in many variants substituting the chlorine with N-hydroxy succinimide (U.S. Pat. No. 5,122,614, (1992); Zalipsky et al., (1992), Biotechnol. Appl. Biochem., 15, p. 100-114; Monfardini et al., (1995), Bioconjugate Chem., 6, 62-69, with imidazole (Allen et al., (1991), Carbohydr. Res., 213, pp 309-319), with para-nitrophenol, DMAP (EP 632 082 A1, (1993), Looze, Y.) etc. The derivatives are usually made by reacting the chloroformate with the desired leaving group. All these groups give rise to carbamate linkages to the peptide.

Furthermore, isocyanates and isothiocyanates may be employed, yielding ureas and thioureas, respectively.

Amides may be obtained from PEG acids using the same leaving groups as mentioned above and cyclic imid thrones (U.S. Pat. No. 5,349,001, (1994), Greenwald et al.). The reactivity of these compounds are very high but may make the hydrolysis to fast.

PEG succinate made from reaction with succinic anhydride can also be used. The hereby comprised ester group make the conjugate much more susceptible to hydrolysis (U.S. Pat. No. 5,122,614, (1992), Zalipsky). This group may be activated with N-hydroxy succinimide.

Furthermore, a special linker can be introduced. The most well studied being cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; U.S. Pat. No. 4,179,337, (1979), Davis et al.; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378.

Coupling of PEG to an aromatic amine followed by diazotation yields a very reactive diazonium salt, which can be reacted with a peptide in situ. An amide linkage may also be obtained by reacting an azlactone derivative of PEG (U.S. Pat. No. 5,321,095, (1994), Greenwald, R. B.) thus introducing an additional amide linkage.

As some peptides do not comprise many Lysines it may be advantageous to attach more than one PEG to the same Lysine. This can be done e.g. by the use of 1,3-diamino-2-propanol.

PEGs may also be attached to the amino-groups of the enzyme with carbamate linkages (WO 95/11924, Greenwald et al.). Lysine residues may also be used as the backbone.

The coupling technique used in the examples is the N-succinimidyl carbonate conjugation technique descried in WO 90/13590 (Enzon).

In a preferred embodiment, the activated polymer is methyl-PEG which has been activated by N-succinimidyl carbonate as described WO 90/13590. The coupling can be carried out at alkaline conditions in high yields.

For coupling of polymers to the protein variants, it is preferred to use conditions similar to those described in WO 96/17929 and WO 99/00489 (Novo Nordisk A/S) e.g. mono or bis activated PEG's of molecular weight ranging from 100 to 5000 Da. For instance, a methyl-PEG 350 could be activated with N-succinimidyl carbonate and incubated with protein variant at a molar ratio of more than 5 calculated as equivalents of activated PEG divided by moles of lysines in the protein of interest. For coupling to immobilized protein variant, the PEG:protein ratio should be optimized such that the PEG concentration is low enough for the buffer capacity to maintain alkaline pH throughout the reaction; while the PEG concentration is still high enough to ensure sufficient degree of modification of the protein. Further, it is important that the activated PEG is kept at conditions that prevent hydrolysis (i.e. dissolved in acid or solvents) and diluted directly into the alkaline reaction buffer. It is essential that primary amines are not present other than those occurring in the lysine residues of the protein. This can be secured by washing thoroughly in borate buffer. The reaction is stopped by separating the fluid phase containing unreacted PEG from the solid phase containing protein and derivatized protein. Optionally, the solid phase can then be washed with tris buffer, to block any unreacted sites on PEG chains that might still be present.

Introduction of Consensus Sequences for Post-Translational Modifications in the Epitope Areas In another embodiment, the mutations are designed, such that recognition sites for post-translational modifications are introduced in the epitope areas, and the protein variant is expressed in a suitable host organism capable of the corresponding post-translational modification. These post-translational modifications may serve to shield the epitope and hence lower the immunogenicity of the protein variant relative to the protein backbone. Post-translational modifications include glycosylation, phosphorylation, N-terminal processing, acylation, ribosylation and sulfatation. A good example is N-glycosylation. N-glycosylation is found at sites of the sequence Asn-Xaa-Ser, Asn-Xaa-Thr, or Asn-Xaa-Cys, in which neither the Xaa residue nor the amino acid following the tri-peptide consensus sequence is a proline (T. E. Creighton, ‘Proteins—Structures and Molecular Properties, 2nd edition, W. H. Freeman and Co., New York, 1993, pp. 91-93). It is thus desirable to introduce such recognition sites in the sequence of the backbone protein. The specific nature of the glycosyl chain of the glycosylated protein variant may be linear or branched depending on the protein and the host cells. Another example is phosphorylation: The protein sequence can be modified so as to introduce serine phophorylation sites with the recognition sequence arg-arg-(xaa)_n-ser (where n=0, 1, or 2) (SEQ ID NOS: 38 and 39), which can be phosphorylated by the cAMP-dependent kinase or tyrosine phosphorylation sites with the recognition sequence -lys/arg-(xaa)₃-asp/glu- (xaa)₃-tyr (SEQ ID NO: 40), which can usually be phophorylated by tyrosine-specific kinases (T. E. Creighton, “Proteins—Structures and molecular properties”, 2nd ed., Freeman, N.Y., 1993).

Randomized Approaches to Introduce Modifications in Epitope Areas

In order to generate protein variants, more than one amino acid residue may be substituted, added or deleted, these amino acids preferably being located in different epitope areas. In that case, it may be difficult to assess a priori how well the functionality of the protein is maintained while antigenicity is reduced, especially since the possible number of mutation-combinations becomes very large, even for a small number of mutations. In that case, it will be an advantage, to establish a library of diversified mutants each having one or more changed amino acids introduced and selecting those variants, which show good retention of function and at the same time a significant reduction in antigenicity.

A diversified library can be established by a range of techniques known to the person skilled in the art (Reetz M T; Jaeger K E, in ‘Biocatalysis—from Discovery to Application’ edited by Fessner W D, Vol. 200, pp. 31-57 (1999); Stemmer, Nature, vol. 370, p.389-391, 1994; Zhao and Arnold, Proc. Natl. Acad. Sci., USA, vol. 94, pp. 7997-8000, 1997; or Yano et al., Proc. Natl. Acad. Sci., USA, vol. 95, pp 5511-5515, 1998). These include, but are not limited to, ‘spiked mutagenesis’, in which certain positions of the protein sequence are randomized by carring out PCR mutagenesis using one or more oligonucleotide primers which are synthesized using a mixture of nucleotides for certain positions (Lanio T, Jeltsch A, Biotechniques, Vol. 25(6), 958,962,964-965 (1998)). The mixtures of oligonucleotides used within each triplet can be designed such that the corresponding amino acid of the mutated gene product is randomized within some predetermined distribution function. Algorithms have been disclosed, which facilitate this design (Jensen L J et al., Nucleic Acids Research, Vol. 26(3), 697-702 (1998)).

In an embodiment substitutions are found by a method comprising the following steps: 1) a range of substitutions, additions, and/or deletions are listed encompassing several epitope areas (preferably in the corresponding epitope sequences, anchor amino aids, and/or hot spots), 2) a library is designed which introduces a randomized subset of these changes in the amino acid sequence into the target gene, e.g. by spiked mutagenesis, 3) the library is expressed, and preferred variants are selected. In another embodiment, this method is supplemented with additional rounds of screening and/or family shuffling of hits from the first round of screening (J. E. Ness, et al, Nature Biotechnology, vol. 17, pp. 893-896, 1999) and/or combination with other methods of reducing immunogenicity by genetic means (such as that disclosed in WO 92/10755).

The library may be designed, such that at least one amino acid of the epitope area is substituted. In a preferred embodiment at least one amino acid of the epitope sequence itself is changed, and in an even more preferred embodiment, one or more hot spot amino acids are changed. The library may be biased such that towards introducing an amino acid of different size, hydrophilicity, and/or polarity relative to the original one of the ‘protein backbone’. For example changing a small amino acid to a large amino acid, a hydrophilic amino acid to a hydrophobic amino acid, a polar amino acid to a non-polar amino acid or a basic to an acidic amino acid. Other changes may be the addition or deletion of at least one amino acid of the epitope area, preferably deleting an anchor amino acid. Furthermore, substituting some amino acids and deleting or adding others may change an epitope.

Diversity in the protein variant library can be generated at the DNA triplet level, such that individual codons are variegated e.g. by using primers of partially randomized sequence for a PCR reaction. Further, several techniques have been described, by which one can create a library with such diversity at several locations in the gene, which are too far apart to be covered by a single (spiked) oligonucleotide primer. These techniques include the use of in vivo recombination of the individually diversified gene segments as described in WO 97/07205 on page 3, line 8 to 29 or by using DNA shuffling techniques to create a library of full length genes that combine several gene segments each of which are diversified e.g. by spiked mutagenesis (Stemmer, Nature 370, pp. 389-391, 1994 and U.S. Pat. Nos. 5,605,793 and 5,830,721). In the latter case, one can use the gene encoding the “protein backbone” as a template double-stranded polynucleotide and combining this with one or more single or double-stranded oligonucleotides as described in claim 1 of U.S. Pat. No. 5,830,721. The single-stranded oligonucleotides could be partially randomized during synthesis. The double-stranded oligonucleotides could be PCR products incorporating diversity in a specific region. In both cases, one can dilute the diversity with corresponding segments containing the sequence of the backbone protein in order to limit the number of changes that are on average introduced. As mentioned above, methods have been established for designing the ratios of nucleotides (A; C; T; G) used at a particular codon during primer synthesis, so as to approximate a desired frequency distribution among a set of desired amino acids at that particular codon. This allows one to bias the partially randomized mutagenesis towards e.g. introduction of post-translational modification sites, chemical modification sites, or simply amino acids that are different from those that define the epitope or the epitope area. One could also approximate a sequence in a given location or epitope area to the corresponding location on a homologous, human protein.

Occasionally, one would be interested in testing a library that combines a number of known mutations in different locations in the primary sequence of the ‘protein backbone’. These could be introduced post-translational or chemical modification sites, or they could be mutations, which by themselves had proven beneficial for one reason or another (e.g. decreasing antigenicity, or improving specific activity, performance, stability, or other characteristics). In such cases, it may be desirable to create a library of diverse combinations of known sequences. For example if 12 individual mutations are known, one could combine (at least) 12 segments of the ‘protein backbone’ gene in which each segment is present in two forms: one with and one without the desired mutation. By varying the relative amounts of those segments, one could design a library (of size 2¹²) for which the average number of mutations per gene can be predicted. This can be a useful way of combining elements that by themselves give some, but not sufficient effect, without resorting to very large libraries, as is often the case when using ‘spiked mutagenesis’. Another way to combine these ‘known mutations’ could be by using family shuffling of oligomeric DNA encoding the known changes with fragments of the full length wild type sequence.

Assays for Reduced Allergenicity

When protein variants have been constructed based on the methods described in this invention, it is desirable to confirm their antibody binding capacity, functionality, immunogenicity and/or allergenicity using a purified preparation. For that use, the protein variant of interest can be expressed in larger scale, purified by conventional techniques, and the antibody binding and functionality should be examined in detail using dose-response curves and e.g. direct or competitive ELISA (C-ELISA).

The potentially reduced allergenicity (which is likely, but not necessarily true for a variant w. low antibody binding) should be tested in in vivo or in vitro model systems: e.g. an in vitro assays for immunogenicity such as assays based on cytokine expression profiles or other proliferation or differentiation responses of epithelial and other cells inc. B-cells and T-cells. Further, animal models for testing allergenicity should be set up to test a limited number of protein variants that show desired characteristics in vitro. Useful animal models include the guinea pig intratracheal model (GPIT) (Ritz, et al. Fund. Appl. Toxicol., 21, pp. 31-37, 1993), mouse subcutaneous (mouse-SC) (WO 98/30682, Novo Nordisk), the rat intratracheal (rat-IT) (WO 96/17929, Novo Nordisk), and the mouse intranasal (MINT) (Robinson et al., Fund. Appl. Toxicol. 34, pp. 15-24, 1996) models.

The immunogenicity of the protein variant is measured in animal tests, wherein the animals are immunised with the protein variant and the immune response is measured. Specifically, it is of interest to determine the allergenicity of the protein variants by repeatedly exposing the animals to the protein variant by the intratracheal route and following the specific IgG and IgE titers. Alternatively, the mouse intranasal (MINT) test can be used to assess the allergenicity of protein variants. By the present invention the allergenicity is reduced at least 3 times as compared to the allergenicity of the parent protein, preferably 10 times reduced, more preferably 50 times.

However, the present inventors have demonstrated that the performance in ELISA correlates closely to the immunogenic responses measured in animal tests. To obtain a useful reduction of the allergenicity of a protein, the IgE binding capacity of the protein variant must be reduced to at least below 75%, preferably below 50%, more preferably below 25% of the IgE binding capacity of the parent protein as measured by the performance in IgE ELISA, given the value for the IgE binding capacity of the parent protein is set to 100%.

Thus a first asessment of the immunogenicity and/or allergenicity of a protein can be made by measuring the antibody binding capacity or antigenicity of the protein variant using appropriate antibodies. This approach has also been used in the literature (WO 99/47680).

Assays for Altered Immunotherapeutic Effect

The immunotherapeutic effect of allergen vaccines can be assessed a number of different ways. One is to measure the specific IgE binding, the reduction of which indicates a better allergen vaccine potential (WO 99/47680, ALK-ABELLÓ). Also, several cellular assays could be employed to show the modified immuneresponse indicative of good allergen vaccine potential as shown in several publications, all of which are hereby incorporated by reference (van Neerven et al, “T lymphocyte responses to allergens: Epitope-specificity and clinical relevance”, Immunol Today, 1996, vol. 17, pp. 526-532; Hoffmann et al., Allergy, 1999, vol. 54, pp. 446-454, WO 99/07880).

Eventually, clinical trials with allergic patients could be employed using cellular or clinical end-point measurements. (Ebner et al., Clin. Exp. All., 1997, vol. 27, pp. 107-1015; Int. Arch. Allergy Immunol., 1999, vol. 119, pp 1-5).

Determining Functionality

A wide variety of protein functionality assays are available in the literature. Especially, those suitable for automated analysis are useful for this invention. Several have been published in the literature such as protease assays (WO 99/34011, Genencor International; J. E. Ness, et al, Nature Biotechn., 17, pp. 893-896, 1999), oxidoreductase assays (Cherry et al., Nature Biotechn., 17, pp. 379-384, 1999, and assays for several other enzymes (WO 99/45143, Novo Nordisk). Those assays that employ soluble substrates can be employed for direct analysis of functionality of immobilized protein variants.

Cross-Reactivity

A related objective is to reduce cross-reactivity between ‘commercial allergens’ and ‘environmental allergens’. Cross-reactivities between food allergens of different origin are well-known (Akkerdaas et al, Allergy 50, pp 215-220, 1995). Similarly, cross-reactivities between other environmental allergens (like pollen, dust mites etc.) and commercial allergens (like enzyme proteins) have been established in the literature (J. All. Clin. Immunol., 1998, vol. 102, pp. 679-686 and by the present inventors. The molecular reason for this cross-reactivity can be explored using epitope mapping. By finding epitope patterns using antibodies raised against environmental allergen (donor protein) and mapping this information on a commercial allergen (the acceptor protein), one may find the epitopes that are common to both proteins, and hence responsible for the cross-reactivity. Obviously, one can also use the commercial allergen as donor and the environmental allergen as acceptor. By modifying the commercial allergen using protein engineering in the epitope areas identified as described above, one can reduce the cross-reactivity of the commercial allergen variant towards the environmental allergens (and vice versa). Hence, the use of the modified commercial allergens would be safer than using the unmodified commercial allergen.

Testing of this approach would be done using an antibody-binding assay with the protein variant (and its parent protein as control) and antibodies raised against the protein that cross-reacts with the parent protein. The method is otherwise identical to those described in the Methods section for characterization of allergencitiy and antigenicity.

Wash Performance etc.

The modifications of the enzymes in the epitope areas as disclosed the present application may cause other effects to the enzyme than modified immunogenicity. A modification may also change the performance of the enzyme, such as the wash performance, thermo stability, storage stability and increased catalytical activity of the enzyme.

The ability of an enzyme to catalyze the degradation of various naturally occurring substrates present on the objects to be cleaned during e.g. wash is often referred to as its washing ability, wash-ability, detergency, or wash performance. Throughout this application the term wash performance will be used to encompass this property.

Commercial Enzyme Applications

Industrial Applications

Another aspect of the invention is a composition comprising at least one protein (polypeptide) or enzyme of the invention. The composition may comprise other polypeptides, proteins or enzymes and/or ingredients normally used in personal care products, such as shampoo, soap bars, skin lotion, skin creme, hair dye, toothpaste, household articles, agro chemicals, personal care products, such as cleaning preparations e.g. for contact lenses, cosmetics, toiletries, oral and dermal pharmaceuticals, compositions used for treating textiles, compositions used for manufacturing food, e.g. baking, and feed etc.

Examples of said proteins(polypeptides)/enzymes include enzymes exhibiting protease, lipolytic enzyme, oxidoreductase, carbohydrase, transferase, such as transglutaminase, phytase and/or anti-microbial polypeptide activity. These enzymes may be present as conjugates with reduced activity.

The protein of the invention may furthermore typically be used in detergent composition. It may be included in the detergent composition in the form of a non-dusting granulate, a stabilized liquid, or a protected enzyme. Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with mean molecular weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in patent GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Other enzyme stabilizers are well known in the art. Protected enzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition may be in any convenient form, e.g. as powder, granules, paste or liquid. A liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, each of which may be anionic, nonionic, cationic, or zwitterionic. The detergent will usually contain 0-50% of anionic surfactant such as linear alkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES), secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid, or soap. It may also contain 0-40% of nonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as described in WO 92/06154).

The detergent composition may additionally comprise one or more other enzymes, such as e.g. proteases, amylases, lipolytic enzymes, cutinases, cellulases, peroxidases, oxidases, and further anti-microbial polypeptides.

The detergent may contain 1-65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst). The detergent may also be unbuilt, i.e. essentially free of detergent builder.

The detergent may comprise one or more polymers. Examples are carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP), polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylates such as polyacrylates, maleiclacrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂0₂ source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfon-ate (NOBS). Alternatively, the bleaching system may comprise peroxyacids of, e.g., the amide, imide, or sulfone type.

The detergent composition of the invention comprising the polypeptide of the invention may be stabilized using conventional stabilizing agents, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative such as, e.g., an aromatic borate ester, and the composition may be formulated as described in, e.g., WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredients such as, e.g., fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil-redeposition agents, dyes, bactericides, optical brighteners, or perfume.

The pH (measured in aqueous solution at use concentration) will usually be neutral or alkaline, e.g. in the range of 7-11.

Dishwashing Composition

Further, a modified enzyme according to the invention may also be used in dishwashing detergents.

Dishwashing detergent compositions comprise a surfactant which may be anionic, non-ionic, cationic, amphoteric or a mixture of these types. The detergent will contain 0-90% of non-ionic surfactant such as low- to non-foaming ethoxylated propoxylated straight-chain alcohols.

The detergent composition may contain detergent builder salts of inorganic and/or organic types. The detergent builders may be subdivided into phosphorus-containing and non-phosphorus-containing types. The detergent composition usually contains 1-90% of detergent builders.

Examples of phosphorus-containing inorganic alkaline detergent builders, when present, include the water-soluble salts especially alkali metal pyrophosphates, orthophosphates, and polyphosphates. An example of phosphorus-containing organic alkaline detergent builder, when present, includes the water-soluble salts of phosphonates. Examples of non-phosphorus-containing inorganic builders, when present, include water-soluble alkali metal carbonates, borates and silicates as well as the various types of water-insoluble crystalline or amorphous alumino silicates of which zeolites are the best-known representatives.

Examples of suitable organic builders include the alkali metal, ammonium and substituted ammonium, citrates, succinates, malonates, fatty acid sulphonates, carboxymetoxy succinates, ammonium polyacetates, carboxylates, polycarboxylates, aminopolycarboxylates, polyacetyl carboxylates and polyhydroxsulphonates.

Other suitable organic builders include the higher molecular weight polymers and co-polymers known to have builder properties, for example appropriate polyacrylic acid, polymaleic and polyacrylic/polymaleic acid copolymers and their salts.

The dishwashing detergent composition may contain bleaching agents of the chlorine/bromine-type or the oxygen-type. Examples of inorganic chlorine/bromine-type bleaches are lithium, sodium or calcium hypochlorite and hypobromite as well as chlorinated trisodium phosphate. Examples of organic chlorine/bromine-type bleaches are heterocyclic N-bromo and N-chloro imides such as trichloroisocyanuric, tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids, and salts thereof with water-solubilizing cations such as potassium and sodium. Hydantoin compounds are also suitable.

The oxygen bleaches are preferred, for example in the form of an inorganic persalt, preferably with a bleach precursor or as a peroxy acid compound. Typical examples of suitable peroxy bleach compounds are alkali metal perborates, both tetrahydrates and monohydrates, alkali metal percarbonates, persilicates and perphosphates. Preferred activator materials are TAED and glycerol triacetate.

The dishwashing detergent composition of the invention may be stabilized using conventional stabilizing agents for the enzyme(s), e.g. a polyol such as e.g. propylene glycol, a sugar or a sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g. an aromatic borate ester.

The dishwashing detergent composition of the invention may also contain other conventional detergent ingredients, e.g. deflocculant material, filler material, foam depressors, anti-corrosion agents, soil-suspending agents, sequestering agents, anti-soil redeposition agents, dehydrating agents, dyes, bactericides, fluorescers, thickeners and perfumes.

Finally, the enzyme of the invention may be used in conventional dishwashing-detergents, e.g. in any of the detergents described in any of the following patent publications: EP 518719, EP 518720, EP 518721, EP 516553, EP 516554, EP 516555, GB 2200132, DE 3741617, DE 3727911, DE 4212166, DE 4137470, DE 3833047, WO 93/17089, DE 4205071, WO 52/09680, WO 93/18129, WO 93/04153, WO 92/06157, WO 92/08777, EP 429124, WO 93/21299, U.S. Pat. No. 5,141,664, EP 561452, EP 561446, GB 2234980, WO 93/03129, EP 481547, EP 530870, EP 533239, EP 554943, EP 346137, U.S. Pat. No. 5,112,518, EP 318204, EP 318279, EP 271155, EP 271156, EP 346136, GB 2228945, CA 2006687, WO 93/25651, EP 530635, EP 414197, and U.S. Pat. No. 5,240,632.

Personal Care Applications

A particularly useful application area for low allergenic proteins or of proteins with low cross-reactivity to environmental allergens would be in personal care products where the end-user is in close contact with the protein, and where certain problems with allergenicity has been encountered in experimental set-ups (Kelling et al., J. All. Clin. Imm., 1998, Vol.101, pp. 179-187 and Johnston et al., Hum. Exp. Toxicol., 1999, Vol. 18, p. 527).

First of all the conjugate or compositions of the invention can advantageously be used for personal care products, such as hair care and hair treatment products. This include products such as shampoo, balsam, hair conditioners, hair waving compositions, hair dyeing compositions, hair tonic, hair liquid, hair cream, shampoo, hair rinse, hair spray.

Further contemplated are oral care products such as dentifrice, oral washes, chewing gum.

Also contemplated are skin care products and cosmetics, such as skin cream, skin milk, cleansing cream, cleansing lotion, cleansing milk, cold cream, cream soap, nourishing essence, skin lotion, milky lotion, calamine lotion, hand cream, powder soap, transparent soap, sun oil, sun screen, shaving foam, shaving cream, baby oil lipstick, lip cream, creamy foundation, face powder, powder eye-shadow, powder, foundation, make-up base, essence powder, whitening powder.

Also for contact lenses hygiene products the conjugate of the invention can be used advantageously. Such products include cleaning and disinfection products for contact lenses.

Proteases

Proteases are well-known active ingredients for cleaning of contact lenses. They hydrolyse the proteinaceous soil on the lens and thereby makes it soluble. Removal of the protein soil is essential for the wearing comfort.

Proteases are also effective ingredients in skin cleaning products, where they remove the upper layer of dead keratinaseous skin cells and thereby make the skin look brighter and fresher.

Proteases are also used in oral care products, especially for cleaning of dentures, but also in dentifrices.

Further, proteases are used in toiletries, bath and shower products, including shampoos, conditioners, lotions, creams, soap bars, toilet soaps, and liquid soaps.

Lipolytic Enzymes

Lipolytic enzymes can be applied for cosmetic use as active ingredients in skin cleaning products and anti-acne products for removal of excessive skin lipids, and in bath and shower products such as creams and lotions as active ingredients for skin care.

Lipolytic enzymes can also be used in hair cleaning products (e.g. shampoos) for effective removal of sebum and other fatty material from the surface of hair.

Lipolytic enzymes are also effective ingredients in products for cleaning of contact lenses, where they remove lipid deposits from the lens surface.

Oxidoreductases

The most common oxidoreductase for personal care purposes is an oxidase (usually glucose oxidase) with substrate (e.g. glucose) that ensures production of H₂O₂, which then will initiate the oxidation of for instance SCN⁻ or I⁻ into antimicrobial reagents (SCNO⁻ or I₂) by a peroxidase (usually lactoperoxidase). This enzymatic complex is known in nature from e.g. milk and saliva.

It is being utilised commercially as anti-microbial system in oral care products (mouth rinse, dentifrice, chewing gum) where it also can be combined with an amyloglucosidase to produce the glucose. These systems are also known in cosmetic products for preservation.

Anti-microbial systems comprising the combination of an oxidase and a peroxidase are know in the cleaning of contact lenses.

Another application of oxidoreductases is oxidative hair dyeing using oxidases, peroxidases and laccases.

Free radicals formed on the surface of the skin (and hair) known to be associated with the ageing process of the skin (spoilage of the hair). The free radicals activate chain reactions that lead to destruction of fatty membranes, collagen, and cells. The application of free radical scavengers such as Superoxide dismutase into cosmetics is well known (R. L. Goldemberg, DCI, Nov. 93, p. 48-52).

Protein disulfide isomerase (PDI) is also an oxidoreductase. It can be utilised for waving of hair (reduction and reoxidation of disulfide bonds in hair) and repair of spoiled hair (where the damage is mainly reduction of existing disulfide bonds).

Carbohydrases

Plaque formed on the surface of teeth is composed mainly of polysaccharides. They stick to the surface of the teeth and the microorganisms. The polysaccharides are mainly α-1,6 bound glucose (dextran) and α-1,3 bound glucose (mutan). The application of different types of glucanases such as mutanase and dextranase helps hydrolysing the sticky matrix of plaque, making it easier to remove by mechanical action.

Also other kinds of biofilm for instance the biofilm formed in lens cases can be removed by the action of glucanases.

Food and Feed

Further conjugated enzymes or polypeptides with reduced immunogenicity according to the invention may advantageously be used in the manufacturing of food and feed.

Proteases

The gluten in wheat flour is the essential ingredient responsible for the ability of flour to be used in baked foodstuffs. Proteolytic enzymes are sometimes needed to modify the gluten phase of the dough, e.g. a hard wheat flour can be softened with a protease.

Neutrase® is a commercially available neutral metallo protease that can be used to ensure a uniform dough quality and bread texture, and to improve flavour. The gluten proteins are degraded either moderately or more extensively to peptides, whereby close control is necessary in order to avoid excessive softening of the dough.

Proteases are also used for modifying milk protein.

To coagulate casein in milk when producing cheese proteases such as rennet or chymosin may be used.

In the brewery industry proteases are used for brewing with unmalted cereals and for controlling the nitrogen content.

In animal feed products proteases are used so to speak to expand the animals digestion system.

Lipolytic Enzymes

Addition of lipolytic enzyme results in improved dough properties and an improved breadmaking quality in terms of larger volume, improved crumb structure and whiter crumb colour. The observed effect can be explained by a mechanism where the lipolytic enzyme changes the interaction between gluten and some lipids fragment during dough mixing. This results in an improved gluten network.

The flavour development of blue roan cheese (e.g. Danablue), certain Italian type cheese, and other dairy products containing butter-fat, are dependent on the degradation of milk fat into free fatty acids. Lipolytic enzymes may be used for developing flavour in such products.

In the oil- and fat producing industry lipases are used e.g. to minimize the amount of undesirable side-products, to modify fats by interesterification, and to synthesis of esters.

Oxidoreductases

Further oxidoreductases with reduced immunogenicity according to the invention may advantageously be used in the manufacturing of food and feed.

Several oxidoreductases are used for baking, glucose oxidase, lipoxygenase, peroxidase, catalase and combinations hereof. Traditionally, bakers strengthen gluten by adding ascorbic acid and potassium bromate. Some oxidoreductases can be used to replace bromate in dough systems by oxidation of free sulfydryl units in gluten proteins. Hereby disulphide linkages are formed resulting in stronger, more elastic doughs with greater resistance.

Gluzyme™ (Novozymes A/S) is a glucose oxidase preparation with catalase activity that can be used to replace bromate. The dough strengthen is measured as greater resistance to mechanical shock, better oven spring and larger loaf volume.

Carbohydrases

Flour has varying content of amylases leading to differences in the baking quality. Addition of amylases can be necessary in order to standardize the flour. Amylases and pentosanases generally provide sugar for the yeast fermentation, improve the bread volume, retard retrogradation, and decrease the staling rate and stickiness that results from pentosan gums.

Examples of Carbohydrases Are Given Below

Certain maltogenic amylases can be used for prolonging the shelf life of bread for two or more days without causing gumminess in the product. Selectively modifies the gelatinized starch by cleaving from the non-reducing end of the starch molecules, low molecular wight sugars and dextrins. The starch is modified in such a way that retrogradation is less likely to occur. The produced low-molecular-weight sugars improve the baked goods water retention capacity without creating the intermediate-length dextrins that result in gumminess in the finished product. The enzyme is inactivated during bread baking, so it can be considered a processing aid that does not have to be declared on the label. Overdosing of Novamyl can almost be excluded. is The bread volume can be improved by fungal α-amylases which further provide good and uniform structure of the bread crumb. Said α-amylases are endoenzymes that produce maltose, dextrins and glucose. Cereal and some bacterial α-amylases are inactivated at temperatures above the gelatinization temperature of starch, therefore when added to wheat dough it results in a low bread volume and a sticky bread interior. Fungamyl has the advantage of being thermolabile and is inactivated just below the gelatinization temperature.

Enzyme preparations containing a number of pentosanase and hemi-cellulase activities can improve the handling and stability of the dough, and improves the freshness, the crumb structure and the volume of the bread.

By hydrolysing the pentosans fraction in flour, it will lose a great deal of its water-binding capacity, and the water will then be available for starch and gluten. The gluten becomes more pliable and extensible, and the starch gelatinizes more easily. Pentosanases can be used in combination with or as an alternative to emulsifiers.

Further carbohydrases are user for producing syrups from starch, which are widely used in soft drinks, sweets, meat products, dairy products, bread products, ice cream, baby food, jam etc.

The conversion of starch is normally carried out three steps. First the starch is liquefied, by the use of alpha-amylases. Maltodextrins, primary consisting of oligosaccharides and dextrins, are obtained.

The mixture is then treated with an amyloglucosidase for hydrolysing the oligosaccharides and dextrins into glucose. This way a sweeter product is obtained. If high maltose syrups are desired beta-amylases alone or in combination with a pullulanase (de-branching enzyme) may be used.

The glucose mixture can be made even sweeter by isomerization to fructose. For this an immobilized glucose isomerase can be used.

In the sugar industry, it is common practice to speed up the break down of present starch in cane juices. Thereby the starch content in the raw sugar is reduced and filtration at the refinery facilitated.

Furthermore dextranases are used to break down dextran in raw sugar juices and syrups.

In the alcohol industry alpha-amylases is advantageously being used for thinning of starch in distilling mashes.

In the brewing industry alpha-amylases is used for adjunct liquefaction.

In the dairy industry beta-galactosidases (lactase) is used when producing low lactose milk for persons suffering from lactose malabsorption.

When flavoured milk drinks are produced from lactase-treated milk, the addition of sugar can be reduced without reducing the sweetness of the product.

In the production of condensed milk, lactose crystallization can be avoided by lactase treatment, and the risk of thickening caused by casein coagulation in lactose crystals is thus reduced.

When producing ice cream made from lactase-treated milk (or whey) no lactose crystals will be formed and the defect, sandiness, will not occur.

Further, xylanases are known to be used within a number of food/feed industrial applications as described in WO 94/21785 (Novo Nordisk A/S).

Alpha-amylases are used in the animal feed industry to be added to cereal-containing feed to improve the digestibility of starch.

Anti-Microbial Polypeptides

Certain bacteriolytic enzymes may be used e.g. to wash carcasses in the meat packing industry (see U.S. Pat. No. 5,354,681 from Novo Industri A/S)

Transferases

Transglutaminases with reduced immunogenicity according to the invention may advantageously be used in the manufacturing of food and feed.

Transglutaminases has the ability to crosslinking protein.

This property can be used for gelling of aqueous phases containing proteins. This may be used for when producing of spreads (DK patent application no. 1071/84 from Novo Nordisk A/S).

Transglutaminases are being used for improvement of baking quality of flour e.g. by modifying wheat flour to be used in the preparation of cakes with improved properties, such as improved taste, dent, mouth-feel and a higher volume (see JP 1-110147).

Further producing paste type food material e.g. used as fat substitution in foods as ice cream, toppings, frozen desserts, mayonnaises and low fat spreads (see WO 93/22930 from Novo Nordisk A/S).

Furthermore for preparation of gels for yoghurt, mousses, cheese, puddings, orange juice, from milk and milk-like products, and binding of chopped meat product, improvement of taste and texture of food proteins (see WO 94/21120 and WO 94/21129 from Novo Nordisk A/S).

Phytases

Phytases of the invention may advantageously be used in the manufacturing of food, such as breakfast cereal, cake, sweets, drinks, bread or soup etc., and animal feed.

Phytases may be used either for exploiting the phosphorus bound in the phytate/phytic acid present in vegetable protein sources or for exploiting the nutritionally important minerals bound in phytic acid complexes.

Microbial phytase may be added to feedstuff of monogastric animals in order to avoid supplementing the feed with inorganic phosphorus (see U.S. Pat. No. 3,297,548).

Further phytases may be used in soy processing. Soyabean meal may contain high levels of the anti-nutritional factor phytate which renders this protein source unsuitable for application in baby food and feed for fish, calves and other non-ruminants, since the phytate chelates essential minerals present therein (see EP 0 420 358).

Also for baking purposes phytases may be used. Bread with better quality can be prepared by baking divided pieces of a dough containing wheat flour etc. and phytase (see JP-0-3076529-A).

A high phytase activity as in koji mold are known to be used for producing refined sake (see JP-0-6070749-A).

Textile Applications

Proteases

Proteases are used for degumming and sand washing of silk.

Lipolytic Enzymes

Lipolytic enzymes are used for removing fatty matter containing hydrophobic esters (e.g. triglycerides) during the finishing of textiles (see e.g. WO 93/13256 from Novo Nordisk A/S).

Oxidoreductases

In bleach clean up of textiles catalases may serve to remove excess hydrogen peroxide.

Carbohydrases

Cellulolytic enzymes are widely used in the finishing of denim garments in order to provide a localized variation in the colour density of the fabric (Enzyme facilitated “stone wash”).

Also cellulolytic enzymes find use in the bio-polishing process. Bio-Polishing is a specific treatment of the yarn surface which improves fabric quality with respect to handle and appearance without loss of fabric wettability. Bio-polishing may be obtained by applying the method described e.g. in WO 93/20278.

During the weaving of textiles, the threads are exposed to considerable mechanical strain. In order to prevent breaking, the threads are usually reinforced by the coating (sizing) with a gelatinous substance (size). The most common sizing agent is starch in native or modified form. A uniform and durable finish can thus be obtained only after removal of the size from the fabric, the so-called desizing. Desizing of fabrics sized with a size containing starch or modified starch is preferably facilitated by use of amylolytic enzymes.

Oral and Dermal Pharmaceuticals

Proteases

Different combinations of highly purified proteases (e.g. Trypsin and Chymotrypsin) are used in pharmaceuticals to be taken orally, and dermal pharmaceuticals for combating e.g inflammations, edemata and injuries.

Leather Production

Transferase

Transglutaminase is known to be used to casein-finishing leather by acting as a hardening agent (see WO 94/13839 from Novo Nordisk).

Hard Surface Cleaning

Cleaning of hard surfaces e.g. in the food industry is often difficult, as equipment used for producing dairies, meat, sea food products, beverages etc. often have a complicated shape. The use of surfactant compositions in the form gels and foams comprising enzymes have shown to facilitate and improve hard surface cleaning. Enzymes, which advantageously may be added in such surfactant compositions, are in particular proteases, lipolytic enzymes, amylases and cellulases.

Such hard surface cleaning compositions comprising enzymes may also advantageously be used in the transport sector, for instance for washing cars and for general vessel wash.

Furthermore this invention relates to the method by which the protein variants are being synthesised and expressed in host cells. This is achieved by culturing host cells capable of expressing a polypeptide in a suitable culture medium to obtain expression and secretion of the polypeptide into the medium, followed by isolation of the polypeptide from the culture medium. The host cell may be any cell suitable for the large-scale production of proteins, capable of expressing a protein and being transformed by an expression vector.

The host cell comprises a DNA construct as defined above, optionally the cells may be transformed with an expression vector comprising a DNA construct as defined above. The host cell is selected from any suitable cell, such as a bacterial cell, a fungal cell, an animal cell, such as an insect cell or a mammalian cell, or a plant cell.

Immunotherapy

A number of vaccination approaches have been described to for infective diseases as well as for non-infective diseases (such as cancers). In a number of cases, the antigen provided is an isolated protein or protein-adjuvant mixture and more and more often, the protein is recombinant (e.g. the hepatitits B vaccine from Merck & Co). In these cases, it could be desirable to modify the immunogenicity of the antigen vaccine, such that it offers a stronger or more specific protection. This can be achieved by protein engineering of the amino acid sequence of the antigen, and would be greatly facilitated by the use of the methods of this invention for identification of epitopes on the antigen vaccine to be the favored sites for modification.

There are several examples of vaccine molecules that have been engineered to achieve a specific immune protection against virus, parasites or cancer (Ryu and Nam, Biotechnol. Prog., 2000, vol. 16 pp. 2-16; and references cited therein). “The goal is often to vaccinate with a minimal strucutre consisting of a well-defined antigen, to stimulate an effective specific immune response, while avoiding potentially hazardous risks” (Ryu and Nam, Biotechnol. Prog., 2000, vol. 16 pp. 2-16). Thus, the methods of this invention can be used to identify such minimal structures that define an antigen (or epitope thereof) whether in the form of the parent protein scaffold with a number of mutations introduced in it, or whether it is in the form of the antibody binding peptides themselves.

Allergen Vaccines

Today, a patient suffering allergic disease may be subjected to allergy vaccine therapy using allergens selected on the basis of testing the specificity of the patient's serum IgE against a bank of allergen extracts (or similar specificity tests of the patient's sensibilization such as skin prick test.

One could improve the quality of characterization by using antibody binding peptides corresponding to various epitope sequences on the protein allergens of interest. This would require a kit comprising reagents for such specificity characterization, e.g. the antibody binding is peptides of desired specificity. It would be preferred to use antibody binding sequences in the kit, which correspond to defined epitope sequences known to be specific for the allergen under investigation (i.e. not identified on other allergens and/or not cross-reacting with sera raised against other allergens). This kit would be useful to specifying which allergy the patient is suffering from. This kit will lead to a more specific answer than those kits used today, and hence to a better selection of allergen vaccine therapy for the individual patient.

Further, the knowledge about cross-reacting epitopes may improve vaccine development.

In an extension of this approach, one could also characterize the patient's serum by identifying the corresponding antibody binding peptides among a random display library using the aforementioned methods. This again may lead to a better selection of allergen vaccine therapy.

Further, one could use the individual antibody binding sequences as allergen vaccines leading to more specific allergen vaccine. These antibody binding sequences could be administered in an isolated form or fused to a membrane protein of the phage display system, or to another protein, which may have beneficial effect for the immunoprotective effect of the antibody binding peptide (Dalum et al., Nature Biotechnology, 1999, Vol. 17, pp. 666-669).

D) Variations Possible

Parent Protein

The “parent protein” can in principle be any protein molecule of biological origin, non-limiting examples of which are peptides, polypeptides, proteins, enzymes, post-translationally modified polypeptides such as lipopeptides or glycosylated peptides, anti-microbial peptides or molecules, and proteins having pharmaceutical properties etc.

Accordingly the invention relates to a method, wherein the “parent protein” is chosen from the group consisting of polypeptides, small peptides, lipopeptides, antimicrobials, and pharmaceutical polypeptides.

The term “pharmaceutical polypeptides” is defined as polypeptides, including peptides, such as peptide hormones, proteins and/or enzymes, being physiologically active when introduced into the circulatory system of the body of humans and/or animals.

Pharmaceutical polypeptides are potentially immunogenic as they are introduced into the circulatory system.

Examples of “pharmaceutical polypeptides” contemplated according to the invention include insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalmic releasing factors, antidiuretic hormones, thyroid stimulating hormone, relaxin, interferon, thrombopoietin (TPO) and prolactin.

However, the proteins are preferably to be used in industry, housekeeping and/or medicine, such as proteins used in personal care products (for example shampoo; soap; skin, hand and face lotions; skin, hand and face cremes; hair dyes; toothpaste), food (for example in the baking industry), detergents and pharmaceuticals.

Antimicrobial Peptides

The antimicrobial peptide (AMP) may be, e.g., a membrane-active antimicrobial peptide, or an antimicrobial peptide affecting/interacting with intracellular targets, e.g. binding to cell DNA. The AMP is generally a relatively short peptide, consisting of less than 100 amino acid residues, typically 20-80 residues. The antimicrobial peptide has bactericidal and/or fungicidal effect, and it may also have antiviral or antitumour effects. It generally has low cytotoxicity against normal mammalian cells.

The antimicrobial peptide is generally highly cationic and hydrophobic. It typically contains several arginine and lysine residues, and it may not contain a single glutamate or asparatate. It usually contains a large proportion of hydrophobic residues. The peptide generally has an amphiphilic structure, with one surface being highly positive and the other hydrophobic.

The bioactive peptide and the encoding nucleotide sequence may be derived from plants, invertebrates, insects, amphibians and mammals, or from microorganisms such as bacteria and fungi.

The antimicrobial peptide may act on cell membranes of target microorganisms, e.g. through nonspecific binding to the membrane, usually in a membrane-parallel orientation, interacting only with one face of the bilayer.

The antimicrobial peptide typically has a structure belonging to one of five major classes: a helical, cystine-rich (defensin-like), b-sheet, peptides with an unusual composition of regular amino acids, and peptides containing uncommon modified amino acids.

Examples of alpha-helical peptides are Magainin 1 and 2; Cecropin A, B and P1; CAP18; Andropin; Clavanin A or AK; Styelin D and C; and Buforin II. Examples of cystine-rich peptides are a-Defensin HNP-1 (human neutrophil peptide) HNP-2 and HNP-3; b-Defensin-12, Drosomycin, g1-purothionin, and Insect defensin A. Examples of b-sheet peptides are Lactoferricin B, Tachyplesin I, and Protegrin PG1-5. Examples of peptides with an unusual composition are Indolicidin; PR-39; Bactenicin Bac5 and Bac7; and Histatin 5. Examples of peptides with unusual amino acids are Nisin, Gramicidin A, and Alamethicin.

Another example is the antifungal peptide (AFP) from Aspergillus giganteus. As explained in detail in WO 94/01459, which is hereby incorporated by reference, the antifungal polypeptide having the amino acid sequence shown in FIG. 1 has been found in several strains of the fungal species A. giganteus, an example of which is the A. giganteus strain deposited with the Centraallbureau voor Schimmelcultures (CBS) under the deposition number CBS 526.65.

However, the antifungal polypeptide, or variants thereof, suitable for the use according to the invention are expected to be derivable from other fungal species, especially other Aspergillus species such as A. pallidus, A. clavatus, A. longivesica, A. rhizopodus and A. clavatonanicus, because of the close relationship which exists between these species and A. giganteus.

In one embodiment of the invention the protein is an enzyme, such as glycosyl hydrolases, carbohydrases, peroxidases, proteases, lipolytic enzymes, phytases, polysaccharide lyases, oxidoreductases, transglutaminases and glycoseisomerases, in particular the following.

Parent Proteases

Parent proteases (i.e. enzymes classified under the Enzyme Classification number E.C. 3.4 in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) include proteases within this group.

Examples include proteases selected from those classified under the Enzyme Classification (E.C.) numbers:

3.4.11 (i.e. so-called aminopeptidases), including 3.4.11.5 (Prolyl aminopeptidase), 3.4.11.9 (X-pro aminopeptidase), 3.4.11.10 (Bacterial leucyl aminopeptidase), 3.4.11.12 (Thermophilic aminopeptidase), 3.4.11.15 (Lysyl aminopeptidase), 3.4.11.17 (Tryptophanyl aminopeptidase), 3.4.11.18 (Methionyl aminopeptidase).

3.4.21 (i.e. so-called serine endopeptidases), including 3.4.21.1 (Chymotrypsin), 3.4.21.4 (Trypsin), 3.4.21.25 (Cucumisin), 3.4.21.32 (Brachyurin), 3.4.21.48 (Cerevisin) and 3.4.21.62 (Subtilisin);

3.4.22 (i.e. so-called cysteine endopeptidases), including 3.4.22.2 (Papain), 3.4.22.3 (Ficain), 3.4.22.6 (Chymopapain), 3.4.22.7 (Asclepain), 3.4.22.14 (Actinidain), 3.4.22.30 (Caricain) and 3.4.22.31 (Ananain);

3.4.23 (i.e. so-called aspartic endopeptidases), including 3.4.23.1 (Pepsin A), 3.4.23.18 (Aspergillopepsin I), 3.4.23.20 (Penicillopepsin) and 3.4.23.25 (Saccharopepsin); and

3.4.24 (i.e. so-called metalloendopeptidases), including 3.4.24.28 (Bacillolysin).

Serine Proteases

A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 “Principles of Biochemistry,” Fifth Edition, McGraw-Hill Book Company, N.Y., pp. 271-272).

The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Dalton range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711 -753).

Subtilases

A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. They are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously often defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al.(1997).

Savinase-Like Subtilisin

One subgroup of the subtilases may be classified as savinase-like subtilisins, having at least 81% homology to Savinase, preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 96% homology, most preferably at least 98% homology to Savinase.

Parent Subtilase

The term “parent subtilase” describes a subtilase defined according to Siezen et al. (1991 and 1997). For further details see description of “SUBTILASES” immediately above. A parent subtilase may also be a subtilase isolated from a natural source, wherein subsequent modifications have been made while retaining the characteristic of a subtilase. Furthermore, a parent subtilase may also be a subtilase which has been prepared by the DNA shuffling technique, such as described by J. E. Ness et al., Nature Biotechnology, 17, 893-896 (1999).

Alternatively the term “parent subtilase” may be termed “wild type subtilase”.

Modification(s) of a Subtilase Variant

The term “modification(s)” used herein is defined to include chemical modification of a subtilase as well as genetic manipulation of the DNA encoding a subtilase. The modification(s) can be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest.

Subtilase Variant

In the context of this invention, the term subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilase protease is produced when expressed in a suitable host.

Examples of relevant subtilisins comprise subtilisin BPN′, subtilisin amylosacchariticus, subtilisin 168, subtilisin mesentericopeptidase, subtilisin Carlsberg, subtilisin DY, subtilisin 309, subtilisin 147, PD498 (WO 93/24623), thermitase, aqualysin, Bacillus PB92 protease, proteinase K, Protease TW7, and Protease TW3.

Preferred commercially available protease enzymes include Alcalase™, Savinase™, Primase™, Duralase™, Neutrase®, Dyrazym®, Esperase™, Pyrase®, Pancreatic Trypsin NOVO (PTN), Bio-Feed™ Pro, Clear-Lens Pro, and Relase® (Novozymes A/S), Maxatase™, Maxacal™, Maxapem™, Properase™M, Purafect™, Purafect OXP™ (Genencor International Inc.).

It is to be understood that also protease variants are contemplated as the parent protease. Examples of such protease variants are disclosed in EP 130.756 (Genentech), EP 214.435 (Henkel), WO 87/04461 (Amgen), WO 87/05050 (Genex), EP 251.446 (Genencor), EP 260.105 (Genencor), Thomas et al., (1985), Nature. 318, p. 375-376, Thomas et al., (1987), J. Mol. Biol., 193, pp. 803-813, Russel et al., (1987), Nature, 328, p. 496-500, WO 88108028 (Genex), WO 88/08033 (Amgen), WO 89/06279 (Novo Nordisk A/S), WO 91/00345 (Novo Nordisk AIS), EP 525 610 (Solvay) and WO 94/02618 (Gist-Brocades N. V.).

The activity of proteases can be determined as described in “Methods of Enzymatic Analysis”, third edition, 1984, Verlag Chemie, Weinheim, vol. 5.

Parent Lipolytic Enzymes

Lipolytic enzymes are classified in EC 3.1.1 Carboxylic Ester Hydrolases according to Enzyme Nomenclature (available at http://www.chem.qmw.ac.uk/iubmb/enzyme). The lipolytic enzyme may have a substrate specificity with an activity such as EC 3.1.1.3 triacylglycerol lipase, EC 3.1.1.4 phospholipase A2, EC 3.1.1.5 lysophospholipase, EC 3.1.1.26 galactolipase, EC 3.1.1.32 phospholipase A1, EC 3.1.1.73 feruloyl esterase or EC 3.1.1.74 cutinase.

The parent lipolytic enzyme may be prokaryotic, particularly a bacterial enzyme, e.g. from Pseudomonas. Examples are Pseudomonas lipases, e.g. from P. cepacia (U.S. Pat. No. 5,290,694, pdb file 1OIL), P. glumae (N Frenken et al. (1992), Appl. Envir. Microbiol. 58 3787-3791, pdb files 1TAH and 1QGE), P. pseudoalcaligenes (EP 334 462) and Pseudomonas sp. strain SD 705 (FERM BP-4772) (WO 95/06720, EP 721 981, WO 96/27002, EP 812 910). The P. glumae lipase sequence is identical to the amino acid sequence of Chromobacterium viscosum (DE 3908131 A1). Other examples are bacterial cutinases, e.g. from Pseudomonas such as P. mendocina (U.S. Pat. No. 5,389,536) or P. putida (WO 88/09367).

Alternatively, the parent lipolytic enzyme may be eukaryotic, e.g. a fungal lipolytic enzyme such as lipolytic enzymes of the Humicola family and the Zygomycetes family and fungal cutinases.

Examples of fungal cutinases are the cutinases of Fusarium solani pisi (S. Longhi et al., Journal of Molecular Biology, 268 (4), 779-799 (1997)) and Humicola insolens (U.S. Pat. No. 5,827,719).

The parent lipolytic enzyme may be fungal and may have an amino acid sequence that can be aligned with SEQ ID NO: 1 which is the amino acid sequence shown in positions 1-269 of SEQ ID NO: 2 of U.S. Pat. No. 5,869,438 for the lipase from Thermomyces lanuginosus (synonym Humicola lanuginosa), described in EP 258 068 and EP 305 216 (trade name LIPOLASE). The parent lipolytic enzyme may particularly have an amino acid sequence with at least 50% homology with SEQ ID NO: 1. In addition to the lipase from T. lanuginosus, other examples are a lipase from Penicillium camembertii (P25234), a lipase from Fusasrium, lipase/phospholipase from Fusarium oxysporum (EP 130064, WO 98/26057), lipase from F. heterosporum (R87979), lysophospholipase from Aspergillus foetidus (W33009), phospholipase A1 from A. oryzae (JP-A 10-155493), lipase from A. oryzae (D85895), lipase/ferulic acid esterase from A. niger (Y09330), lipase/ferulic acid esterase from A. tubingensis (Y09331), lipase from A. tubingensis (WO 98/45453), lysophospholipase from A. niger (WO 98/31790), lipase from F. solanii having an isoelectric point of 6.9 and an apparent molecular weight of 30 kDa (WO 96/18729).

Other examples are the Zygomycetes family of lipases comprising lipases having at least 50% homology with the lipase of Rhizomucor miehei (P19515. This family also includes the lipases from Absidia reflexa, A. sporophora, A. corymbifera, A. blakesleeana, A. griseola (all described in WO 96/13578 and WO 97/27276) and Rhizopus oryzae (P21811). Numbers in parentheses indicate publication or accession to the EMBL, GenBank, GeneSeqp or Swiss-Prot databases.

Examples of lipases include lipases derived from the following microorganisms. The indicated patent publications are incorporated herein by reference:

Humicola, e.g. H. brevispora, H. brevis var. thermoidea.

Pseudomonas, e.g. Ps. fragi, Ps. stutzeri, Ps. cepacia and Ps. fluorescens (WO 89/04361), or Ps. plantarii or Ps. gladioli (U.S. Pat. No. 4,950,417 (Solvay enzymes)) or Ps. alcaligenes and Ps. pseudoalcaligenes (EP 218272) or.

Candida, e.g. C. cylindracea (also called C. rugosa) or C. antarctica (WO 88/02775) or C. antarctica lipase A or B (WO 94/01541 and WO 89/02916).

Geotricum, e.g. G. candidum (Schimada et al., (1989), J. Biochem., 106, 383-388).

Rhizopus, e.g. R. delemar (Hass et al., (1991), Gene 109, 107-113) or R. niveus (Kugimiya et al., (1992) Biosci. Biotech. Biochem 56, 716-719) or R. oryzae.

Bacillus, e.g. B. subtilis (Dartois et al., (1993) Biochemica et Biophysica acta 1131, 253-260) or B. stearothermophilus (JP 64/7744992) or B. pumilus (WO 91/16422).

Specific examples of readily available commercial lipases include Lipolase® (WO 98/35026) Lipolase™ Ultra, Lipozyme®, Palatase®, Novozym® 435, Lecitase® (all available from Novozymes A/S).

Examples of other lipases are Lumafast™, Ps. mendocian lipase from Genencor Int. Inc.; Lipomax™, Ps. pseudoalcaligenes lipase from Gist Brocades/Genencor Int. Inc.; Fusarium solani lipase (cutinase) from Unilever; Bacillus sp. lipase from Solvay enzymes. Other lipases are available from other companies.

It is to be understood that also lipase variants are contemplated as the parent enzyme. Examples of such are described in e.g. WO 93/01285 and WO 95/22615.

The activity of the lipase can be determined as described in “Methods of Enzymatic Analysis”, Third Edition, 1984, Verlag Chemie, Weinhein, vol. 4, or as described in AF 95/5 GB (available on request from Novozymes A/S).

Parent Oxidoreductases

Parent oxidoreductases (i.e. enzymes classified under the Enzyme Classification number E.C. 1 (Oxidoreductases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) include oxidoreductases within this group.

Examples include oxidoreductases selected from those classified under the Enzyme Classification (E.C.) numbers:

Glycerol-3-phosphate dehydrogenase (NAD) (1.1.1.8), Glycerol-3-phosphate dehydrogenase [NAD(P)] (1.1.1.94), Glycerol-3-phosphate 1-dehydrogenase [NADP] (1.1.1.94), Glucose oxidase (1.1.3.4), Hexose oxidase (1.1.3.5), Catechol oxidase (1.1.3.14), Bilirubin oxidase (1.3.3.5), Alanine dehydrogenase (1.4.1.1), Glutamate dehydrogenase (1.4.1.2), Glutamate dehydrogenase [AND(P)] (1.4.1.3), Glutamate dehydrogenase (NADP) (1.4.1.4), L-Amino acid dehydrogenase (1.4.1.5), Serine dehydrogenase (1.4.1.7), Valine dehydrogenase (NADP) (1.4.1.8), Leucine dehydrogenase (1.4.1.9), Glycine dehydrogenase (1.4.1.10), L-Amino-acid oxidase (1.4.3.2.), D-Amino-acid oxidase(1.4.3.3), L-Glutamate oxidase (1.4.3.11), Protein-lysine 6-oxidase (1.4.3.13), L-lysine oxidase (1.4.3.14), L-Aspartate oxidase (1.4.3.16), D-amino-acid dehydrogenase (1.4.99.1), Protein disulfide reductase (1.6.4.4), Thioredoxin reductase (1.6.4.5), Protein disulfide reductase (glutathione) (1.8.4.2), Laccase (1.10.3.2), Catalase (1.11.1.6), Peroxidase (1.11.1.7), Lipoxygenase (1.13.11.12), Superoxide dismutase (1.15.1.1).

Said glucose oxidases may be derived from Aspergillus niger.

Said laccases may be derived from Polyporus pinsitus, Myceliophthora thermophila, Coprinus cinereus, Rhizoctonia solani, Rhizoctonia praticola, Scytalidium thermophilum and Rhus vernicifera. Because of the homology found between the above mentioned laccases (see WO 98/38287), they are considered to belong to the same class of laccases, namely the class of “Coprinus-like laccases”. Accordingly, in the present context, the term “Coptinus-like laccase” is intended to indicate a laccase which, on the amino acid level, displays a homology of at least 50% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 55% and less than 100% to the Copdinus cinereus laccase SEQ ID NO: 3, or at least 60% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 65% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 70% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 75% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 80% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 85% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 90% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, at least 95% and less than 100% or at least 98% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3.

Bilirubin oxidases may be derived from Myrothechecium verrucaria.

The peroxidase may be derived from e.g. Soy bean, Horseradish or Coprinus cinereus.

The protein disulfide reductase may be any of the mentioned in Danish application nos. 768/93, 265/94 and 264/94 (Novo Nordisk A/S), which are hereby incorporated as references, including Protein Disulfide reductases of bovine origin, Protein Disulfide reductases derived from Aspergillus oryzae or Aspergillus niger, and DsbA or DsbC derived from Escherichia coli.

Specific examples of readily available commercial oxidoreductases include Gluzyme™ (enzyme available from Novozymes A/S). However, other oxidoreductases are available from others.

It is to be understood that also variants of oxidoreductases are contemplated as the parent enzyme.

The activity of oxidoreductases can be determined as described in “Methods of Enzymatic Analysis”, third edition, 1984, Verlag Chemie, Weinheim, vol. 3.

Parent Carbohydrases

Parent carbohydrases may be defined as all enzymes capable of breaking down carbohydrate chains (e.g. starches) of especially five and six member ring structures (i.e. enzymes classified under the Enzyme Classification number E.C. 3.2 (glycosidases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)). Also included in the group of carbohydrases according to the invention are enzymes capable of isomerizing carbohydrates e.g. six member ring structures, such as D-glucose to e.g. five member ring structures like D-fructose.

Examples include carbohydrases selected from those classified under the Enzyme Classification (E.C.) numbers: alpha-amylase (3.2.1.1), beta-amylase (3.2.1.2), glucan 1,4-alpha-glucosidase (3.2.1.3), cellulase (3.2.1.4), endo-1,3(4)-beta-glucanase (3.2.1.6), endo-1,4-beta-xylanase (3.2.1.8), dextranase (3.2.1.11), chitinase (3.2.1.14), polygalacturonase (3.2.1.15), lysozyme (3.2.1.17), beta-glucosidase (3.2.1.21), alpha-galactosidase (3.2.1.22), beta-galactosidase (3.2.1.23), amylo-1,6-glucosidase (3.2.1.33), xylan 1,4-beta-xylosidase (3.2.1.37), glucan endo-1,3-beta-D-glucosidase (3.2.1.39), alpha-dextrin endo-1,6-glucosidase (3.2.1.41), sucrose alpha-glucosidase (3.2.1.48), iglucan endo-1,3-alpha-glucosidase (3.2.1.59), glucan 1,4-beta-glucosidase (3.2.1.74), glucan endo-1,6-beta-glucosidase (3.2.1.75), arabinan endo-1,5-alpha-arabinosidase (3.2.1.99), lactase (3.2.1.108), chitonanase (3.2.1.132) and xylose isomerase (5.3.1.5).

Examples of relevant carbohydrases include alpha-1,3-glucanases derived from Trichoderma harzfanum; alpha-1,6-glucanases derived from a strain of Paecilomyces; beta-glucanases derived from Bacillus subtilis; beta-glucanases derived from Humicola insolens; beta-glucanases derived from Aspergillus niger, beta-glucanases derived from a strain of Trichoderma; beta-glucanases derived from a strain of Oerskovia xanthineolytica; exo-1,4-alpha-D-glucosidases (glucoamylases) derived from Aspergillus niger, alpha-amylases derived from Bacillus subtilis; alpha-amylases derived from Bacillus amyloliquefaciens; alpha-amylases derived from Bacillus stearothermophilus; alpha-amylases derived from Aspergillus oryzae; alpha-amylases derived from non-pathogenic microorganisms; alpha-galactosidases derived from Aspergillus niger, Pentosanases, xylanases, cellobiases, cellulases, hemi-cellulases derived from Humicola insolens; cellulases derived from Trichoderma reesei; cellulases derived from non-pathogenic mold; pectinases, cellulases, arabinases, hemi-celluloses derived from Aspergillus niger, dextranases derived from Penicillium lilacinum; endo-glucanase derived from non-pathogenic mold; pullulanases derived from Bacillus acidopullyticus; beta-galactosidases derived from Kluyveromyces fragilis; xylanases derived from Trichoderma reesei.

Specific examples of readily available commercial carbohydrases include Alpha-Gal™, Bio-Feed™ Alpha, Bio-Feed™ Beta, Bio-Feed™ Plus, Bio-Feed™ Plus, Novozyme® 188, Carezyme® (SEQ ID NO: 5), Celluclast®, Cellusoft®, Ceremyl®, Citrozym™, Denimax™, Dezyme™, Dextrozyme™, Finizym®, Fungamyl™, Gamanase™, Glucanex®, Lactozym®, Maltogenase™, Pentopan™, Pectinex™, Promozyme®, Pulpzyme™, Novamyl™, Termamyl®, AMG (Amyloglucosidase Novo), Maltogenase®, Sweetzyme®, Aquazym®, Natalase® (SEQ ID NO: 4), SP722, AA560 (all enzymes available from Novozymes A/S). Other carbohydrases are available from other companies.

The parent cellulase is preferably a microbial cellulase. As such, the cellulase may be selected from bacterial cellulases, e.g. Pseudomonas cellulases or Bacillus, such as the Bacillus strains described in U.S. Pat. No. 4,822,516, U.S. Pat. No. 5,045,464 or EP 468 464, or B. lautus (cf. WO 91/10732), cellulases. More preferably, the parent cellulases may be a fungal cellulase, in particular Humicola, Trichoderma, Irpex, Aspergillus, Penicillium, Myceliophthora or Fusarium cellulases. Examples of suitable parent cellulases are described in, e.g. WO 91/17244. Examples of suitable Trichoderma cellulases are those described in T. T. Teeri, Gene 51, 1987, pp. 43-52. Preferably, the parent cellulase is selected from the cellulases classified in family 45, e.g. the enzymes EG B (Pseudomonas fluorescens) and EG V (Humicola insolens), as described in Henrissat, B. et al.: Biochem. J. (1993), 293, p. 781-788.

The Termamyl-Like Alpha-Amylase

It is well known that a number of alpha-amylases produced by Bacillus spp. are highly homologous on the amino acid level. For instance, the B. licheniformis alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 4 of WO 00/29560 (commercially available as Termamyl® has been found to be about 89% homologous with the B. amyloliquefaciens alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 5 of WO 00/29560 and about 79% homologous with the B. stearothermophilus alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 3 of WO 00/29560. Further homologous alpha-amylases include an alpha-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO 95/26397, and the alpha-amylase described by Tsukamoto et al., Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31.

Still further homologous alpha-amylases include the alpha-amylase produced by the B. licheniformis strain described in EP 0252666 (ATCC 27811), and the alpha-amylases identified in WO 91/00353 and WO 94/18314. Other commercial Termamyl-like B. licheniformis alpha-amylases are Optitherm® and Takatherm® (available from Solvay), Maxamyl® (available from Gist-brocades/Genencor), Spezym AA® and Spezyme Delta AA™ (available from Genencor), and Keistase® (available from Daiwa).

Because of the substantial homology found between these alpha-amylases, they are considered to belong to the same class of alpha-amylases, namely the class of “Termamyl-like alpha-amylases”.

Accordingly, in the present context, the term “Termamyl-like alpha-amylase” is intended to indicate an alpha-amylase which, at the amino acid level, exhibits a substantial homology to Termamyl®, i.e., the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 4 (WO 00/29560). In other words, a Termamyl-like alpha-amylase is an alpha-amylase which has the amino acid sequence shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or 8 of WO 00/29560, and the amino acid sequence shown in SEQ ID NO: 1 of WO 95/26397 (the same as the amino acid sequence shown as SEQ ID NO: 7 of WO 00/29560) or in SEQ ID NO: 2 of WO 95/26397 (the same as the amino acid sequence shown as SEQ ID NO: 8 of WO 00/29560) or in Tsukamoto et al., 1988, (which amino acid sequence is shown in SEQ ID NO: 6 of WO 00/29560) or i) which displays at least 60% homology (identity), preferred at least 70%, more preferred at least 75%, even more preferred at least 80%, especially at least 85%, especially preferred at least 90%, especially at least 95%, even especially more preferred at least 97%, especially at least 99% homology with at least one of said amino acid sequences shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or 8 of WO 00/29560 and/or ii) displays immunological cross-reactivity with an antibody raised against one or more of said alpha-amylases, and/or iii) is encoded by a DNA sequence which hybridizes, under the low to very high stringency conditions (said conditions described below) to the DNA sequences encoding the above-specified alpha-amylases which are apparent from SEQ ID NOS: 9, 10, 11, 12, and 32, respectively, of the present application (which encodes the amino acid sequences shown in SEQ ID NOS: 1, 2, 3, 4, and 5 herein, respectively), from SEQ ID NO: 4 of WO 95/26397 (which DNA sequence, together with the stop codon TAA, is shown in SEQ ID NO: 13 herein and encodes the amino acid sequence shown in SEQ ID NO: 8 herein) and from SEQ ID NO: 5 of WO 95/26397 (shown in SEQ ID NO: 14 herein), respectively.

In connection with property i), the “homology” (identity) may be determined by use of any conventional algorithm, preferably by use of the gap progamme from the GCG package version 8 (August 1994) using default values for gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1 (Genetic Computer Group (1991) Programme Manual for the GCG Package, version 8, 575 Science Drive, Madison, Wis., USA 53711).

The parent Termamyl-like alpha-amylase backbone may in an embodiment have an amino acid sequence which has a degree of identity to SEQ ID NO: 4 (WO 00/29560) of at least 65%, preferably at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least about 90%, even more preferably at least 95%, even more preferably at least 97%, and even more preferably at least 99% identity determined as described above.

A structural alignment between Termamyl® (SEQ ID NO: 4) and a Termamyl-like alpha-amylase may be used to identify equivalent/corresponding positions in other Termamyl-like alpha-amylases. One method of obtaining said structural alignment is to use the Pile Up programme from the GCG package using default values of gap penelties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1. Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al., (1987), FEBS LETTERS 224, pp. 149-155) and reverse threading (Huber, T; Torda, AE, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).

Parent Glucoamylases

Parent glucoamylase contemplated according to the present invention include fungal glucoamylases, in particular fungal glucoamylases obtainable from an Aspergillus strain, such as an Aspergillus niger or Aspergillus awamori glucoamylases and variants or mutants thereof, homologous glucoamylases, and further glucoamylases being structurally and/or functionally similar to SEQ ID NO: 2 (WO 00/04136). Specifically contemplated are the Aspergillus niger glucoamylases G1 and G2 disclosed in Boel et al. (1984), “Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs”, EMBO J. 3 (5), p. 1097-1102. The G2 glucoamylase is disclosed in SEQ ID NO: 2 (WO 00/04136). The G1 glucoamylase is disclosed in SEQ ID NO: 13 (WO 00/04136). Another AMG backbone contemplated is Talaromyces emersonii, especially Talaromyces emersonii DSM disclosed in WO 99/28448 (Novo Nordisk).

The homology referred to above of the parent glucoamylase is determined as the degree of identity between two protein sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, p. 443-453). Using Gap with the following settings for polypeptide sequence comparison: Gap creation penalty of 3.0 and Gap extension penalty of 0.1, the mature part of a polypeptide encoded by an analogous DNA sequence of the invention exhibits a degree of identity preferably of at least 60%, such as 70%, at least 80%, at least 90%, more preferably at least 95%, more preferably at least 97%, and most preferably at least 99% with the mature part of the amino acid sequence shown in SEQ ID NO: 2 (WO 00/04136).

Preferably, the parent glucoamylase comprise the amino acid sequences of SEQ ID NO: 2(WO 00/04136); or allelic variants thereof; or fragments thereof that has glucoamylase activity.

A fragment of SEQ ID NO: 2 is a polypeptide which have one or more amino acids deleted from the amino and/or carboxyl terminus of this amino acid sequence. For instance, the AMG G2 (SEQ ID NO: 2) is a fragment of the Aspergillus niger G1 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102) having glucoamylase activity. An allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

It is to be understood that also carbohydrase variants are contemplated as the parent enzyme.

The activity of carbohydrases can be determined as described in “Methods of Enzymatic Analysis”, third edition, 1984, Verlag Chemie, Weinheim, vol. 4.

Parent Transferases

Parent transferases (i.e. enzymes classified under the Enzyme Classification number E.C. 2 in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) include transferases within this group.

The parent transferases may be any transferase in the subgroups of transferases: transferases transferring one-carbon groups (E.C. 2.1); transferases transferring aldehyde or residues (E.C 2.2); acyltransferases (E.C. 2.3); glucosyltransferases (E.C. 2.4); transferases transferring alkyl or aryl groups, other that methyl groups (E.C. 2.5); transferases transferring nitrogeneous groups (2.6).

In a preferred embodiment the parent transferase is a transglutaminase E.C 2.3.2.13 (Protein-glutamine μ-glutamyltransferase).

Transglutaminases are enzymes capable of catalyzing an acyl transfer reaction in which a gamma-carboxyamide group of a peptide-bound glutamine residue is the acyl donor. Primary amino groups in a variety of compounds may function as acyl acceptors with the subsequent formation of monosubstituted gamma-amides of peptide-bound glutamic acid. When the epsilon-amino group of a lysine residue in a peptide-chain serves as the acyl acceptor, the transferases form intramolecular or intermolecular gamma-glutamyl-epsilon-lysyl crosslinks.

Examples of transglutaminases are described in the pending DK patent application no. 990/94 (Novo Nordisk ANS).

The parent transglutaminase may be of human, animal (e.g. bovine) or microbial origin.

Examples of such parent transglutaminases are animal derived Transglutaminase, FXIIIa; microbial transglutaminases derived from Physarum polycephalum (Klein et al., Journal of Bacteriology, Vol. 174, p. 2599-2605); transglutaminases derived from Streptomyces sp., including Streptomyces lavendulae, Streptomyces lydicus (former Streptomyces libani) and Streptoverticillium sp., including Streptoverticillium mobaraense, Streptoverticillium cinnamoneum, and Streptoverticillium griseocarneum (Motoki et al., U.S. Pat. No. 5,156,956; Andou et al., U.S. Pat. No. 5,252,469; Kaempfer et al., Journal of General Microbiology, Vol. 137, p. 1831-1892; Ochi et al., International Journal of Sytematic Bacteriology, Vol. 44, p. 285-292; Andou et al., U.S. Pat. No. 5,252,469; Williams et al., Journal of General Microbiology, Vol. 129, p. 1743-1813).

It is to be understood that also transferase variants are contemplated as the parent enzyme.

The activity of transglutaminases can be determined as described in “Methods of Enzymatic Analysis”, third edition, 1984, Verlag Chemie, Weinheim, vol. 1-10.

Parent Phytases

Parent phytases are included in the group of enzymes classified under the Enzyme Classification number E.C. 3.1.3 (Phosphoric Monoester Hydrolases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)).

Phytases are enzymes produced by microorganisms which catalyse the conversion of phytate to inositol and inorganic phosphorus

Phytase producing microorganisms comprise bacteria such as Bacillus subtilis, Bacillus natto and Pseudomonas; yeasts such as Saccharomyces cerevisiae; and fungi such as Aspergillus niger, Aspergillus ficuum, Aspergillus awamori, Aspergillus oryzae, Aspergillus terreus or Aspergillus nidulans, and various other Aspergillus species).

Examples of parent phytases include phytases selected from those classified under the Enzyme Classification (E.C.) numbers: 3-phytase (3.1.3.8) and 6-phytase (3.1.3.26).

The activity of phytases can be determined as described in “Methods of Enzymatic Analysis”, third edition, 1984, Verlag Chemie, Weinheim, vol. 1-10, or may be measured according to the method described in EP-A1-0 420 358, Example 2 A.

Lyases

Suitable lyases include Polysaccharide lyases: Pectate lyases (4.2.2.2) and pectin lyases (4.2.2.10), such as those from Bacillus licheniformis disclosed in WO 99/27083.

Isomerases

Protein Disulfide Isomerase

Without being limited thereto suitable protein disulfide isomerases include PDls described in WO 95/01425 (Novo Nordisk A/S) and suitable glucose isomerases include those described in Biotechnology Letter, Vol. 20, No 6, June 1998, pp. 553-56.

Contemplated isomerases include xylose/glucose Isomerase (5.3.1.5) including Sweetzyme®.

Environmental Allergens

The environmental allergens that are of interest for epitope mapping include allergens from pollen, dust mites, mammals, venoms, fungi, food items, and other plants.

Pollen, allergens include but are not limited to those of the order Fagales, Oleales, Pinales, Poales, Asterales, and Urticales; including those from Betula, Alnus, Corylus, Carpinus, Olea, Phleum pratense and Artemisia vulgaris, such as Aln g1, Cor a1, Car b1, Cry j1, Amb a1 and a2, Art v1, Par j1, Ole e1, Ave v1, and Bet v1 (WO 99/47680).

Mite allergens include but are not limited to those from Derm. farinae and Derm. pteronys., such as Der f1 and f2, and Der p1 and p2.

From mammals, relevant environmental allergens include but are not limited to those from cat, dog, and horse as well as from dandruff from the hair of those animals, such as Fel d1; Can f1; Equ c1; Equ c2; Equ c3.

Venum allergens include but are not limited to PLA2 from bee venom as well as Apis m1 and m2, Ves g1, g2 and g5, Ves v5 and te Pol and Sol allergens.

Fungal allergens include those from Alternaria alt. and Cladospo. herb. such as Alt a1 and Cla h1.

Food allergens include but are not limited to those from milk (lactoglobulin), egg (ovalbumin), peanuts, hazelnuts, wheat (alpha-amylase inhibitor),

Other plant allergens include latex (hevea brasiliensis).

In addition, a number of proteins of interest for expression in transgenic plants could be useful objects for epitope engineering. If for instance a heterologous enzyme is introduced into a transgenic plant e.g. to increase the nutritional value of food or feed derived from that plant, that enzyme may lead to allergenicity problems in humans or animals ingesting the plant-derived material. Epitope mapping and engineering of such heterologous enzymes or other proteins of transgenic plants may lead to reduction or elimination of this problem. Hence, the methods of this patent are also useful for potentially modifying proteins for heterologous expression in plants and plant cells.

Materials and Methods

Materials

ELISA reagents:

• Horse Radish Peroxidase labelled pig anti-rabbit-Ig (Dako, DK, P217, dilution 1:1000)
• Rat anti-mouse IgE (Serotec MCA419; dilution 1:100)
• Mouse anti-rat IgE (Serotec MCA193; dilution 1:200)
• Biotin-labelled mouse anti-rat IgGl monoclonal antibody (Zymed 03-9140; dilution 1:1000)
• Biotin-labelled rat anti-mouse IgGl monoclonal antibody (Serotec MCA336B; dilution 1:2000)
• Streptavidin-horse radish peroxidase (Kirkegard & Perry 14-30-00; dilution 1:1000).

Buffers and Solutions:
PBS (pH 7.2 (1 liter))

	NaCl	8.00 g
	KCl	0.20 g
	K2HPO4	1.04 g
	KH2PO4	0.32 g

• • Washing buffer PBS, 0.05% (v/v) Tween 20
  • Blocking buffer PBS, 2% (wt/v) Skim Milk powder
  • Dilution buffer PBS, 0.05% (v/v) Tween 20, 0.5% (wt/v) Skim Milk powder
  • Citrate buffer 0.1 M, pH 5.0-5.2
  • Stop-solution (DMG-buffer)
  • Sodium Borate, borax (Sigma)
  • 3,3-Dimethyl glutaric acid (Sigma)
  • Tween 20: Poly oxyethylene sorbitan mono laurate (Merck cat no. 822184)
  • PMSF (phenyl methyl sulfonyl flouride) from Sigma
  • Succinyl-Alanine-Alanine-Proline-Phenylalanine-paranitro-anilide (Suc-MPF-pNP) Sigma no. S-7388, Mw 624.6 g/mol.
  • mPEG (Fluka)
    Coloring substrate:

OPD: o-phenylene-diamine, (Kementec cat no. 4260)

Methods

Automatic epitope mapping

Implementation

The implementation consists of 3 pieces of code:

• 1. The core program (see above), written in C (see Appendix A).
• 2. A “wrapping” cgi-script run by the web server, written in Python (see Appendix B).
• 3. A HTML page defining the input/submission form (see Appendix C).

The wrapper receives the input and calls the core program and several other utilities. Apart from the standard Unix utility programs (mv, rm, awk, etc..) the following must be installed:

• • A web server capable of running cgi-scripts, eg. Apache
  • Python 1.5 or later
  • Gnuplot 3.7 or later
  • DSSP, version July 1995
    The core program
    Inputs
• 1. A Brookhaven PDB file with the structure of the protein
• 2. The output of DSSP called with the above PDB file.
• 3. Maximum distance between adjacent residues
• 4. Minimum solvent accessible surface area for each residue
• 5. Maximum epitope size (max distance between any two residues in epitope)
• 6. Maximum number of non-redundant epitopes to include (0=all)
• 7. The shortest acceptable epitope (as a fraction of the length of the epitope consensus sequence).
• 8. Epitope consensus sequence describing which residues are possible at the different positions. An example is shown below:
  • KR (Lys or Arg allowed)
  • AILV- (Ala, lie, Leu, Val or missing residue allowed)
  • * (All residues allowed, but there must be a residue)
  • ? (All or missing residue allowed)
  • DE (Asp or Glu allowed)
  • (*, ? or −in first or last position is allowed but obsolete. (−in first position is ignored.))

Examples of matching epitopes: KAAKD (SEQ ID NO: 41), KLASD (SEQ ID NO: 42), KLYSD (SEQ ID NO: 43), KLY-D (SEQ ID NO: 44), R-M-D.

The Epitope Searching Algorithm

The “core” of the program is the algorithm that scans the protein surface for the epitope patterns. The principle is that several “trees” are built, where each of their branches describes one epitope:

• • 1. All residues in the protein are checked according to: a) Does the residue type match the first residue of the epitope consensus sequence. b) Is the surface accessibility greater than or equal to the given threshold. If both requirements are fulfilled, the protein residue is considered as one root in the epitope tree. Remark that there are usually many roots.
  • 2. For each of the residues defined as roots, all residues within the the given threshold distance between adjacent residues (e.g. 7 Angstroms) are checked for the same as above: a) Does the residue type match the second residue of the epitope consensus sequence. b) Is the surface accessibility greater than or equal to the given threshold. If yes, the protein residue is considered as a “child” of the root. The spatial position of a residue is defined as the coordinates of its C-alpha atom.
  • 3. The procedure from step 2 is repeated for the next residue in the epitope consensus sequence, where each of the “childs” found in step 2 are now “roots” of new childs. If a gap is defined in the epitope consensus sequence, a “missing” residue is allowed, and the coordinates of the root (also called “parent”) is used.
  • 4. This procedure is repeated for all residues in the epitope consensus sequence.
  • 5. In this way a number of trees (corresponding to the number of roots found in step 1) are found. Notice that the same protein residue can be present many places in the trees.
  • 6. If no epitopes that matches the length of the epitope consensus sequence are found, the longest shorter epitopes that matches the first n residues of the epitope consensus sequence are used, where n is an integer smaller than the length of the epitope consensus sequence. If n is smaller than the length of the epitope consensus sequence multiplied by the fraction value defining the shortest acceptable epitope length, no epitopes are written to the output, and steps 7, 8 and 9 are skipped.
  • 7. The epitopes are extracted from the trees by traversing down from each of the “childs” in the last level. The algorithm also finds epitopes which have the same protein residue present more than once. This is, of course, an artifact and such epitopes are discarded. Every epitope is then checked for its size, that is, the maximum distance between any two residues which are members of the epitope. If this exceeds the threshold, the epitope is discarded.
  • 8. Redundant epitopes are removed. Epitopes containing one or more gaps are redundant if they are subsets of other epitopes without or with fewer gaps. For example: A82-gap-F45-G44-K43 is a subset of A82-L46-F45-G44-K43, and is therefore discarded.
  • 9. For every epitope, the total solvent accessible surface area is calculated (by adding the contributions from each residue as found by the DSSP program). The epitopes are sorted according to this area in descending order. If a maximum number of n non-redundant epitopes has been specified, the n epitopes with largest solvent accessible surface area are selected.
  • 10. The output consists of a list of the found epitopes, along with information of the epitope consensus sequence used and other internal parameters. A separate file containing the number of epitopes that each of the protein residues is a member of is also written.
    The wrapper
    Inputs
  • 1. One PDB file, describing one structure, or one ZIP file, containing a number of PDB files, each describing one structure. The ZIP file must not contain subfolders.
  • 2. An epitope consensus sequence or which part of the current epitope library to use (full library or IgE part or IgG part).
  • 3. Maximum distance between adjacent residues
  • 4. Minimum solvent accessible surface area for each residue
  • 5. Maximum epitope size (max distance between any two residues in epitope)
  • 6. Maximum number of non-redundant epitopes to include (0=all)
  • 7. Whether to use sequential numbering (1,2,3,4 . . . etc) or PDB-file numbering.
    Description

The core program accepts only one structure and one epitope consensus sequence. It is usually desirable to use a library of epitope consensus sequences and sometimes several protein structures. The wrapper reads the user input and calls the utility programs and the core program the necessary number of times. The output is collected and presented on the web page returned to the user.

Depending on the type of input, the wrapper works in different modes:

• • Epitope consensus can be given directly or taken from a library
  • Input type can be a single PDB file or a collection of PDB file given as a ZIP-file.

Any of the four possible combinations are allowed.

The epitope library consists of a number of text files, each containing one epitope consensus sequence as specified above.

The layout of the wrapper is like this:

• • 1. Check if the program is already in use from somewhere else (this is done by checking for a lock file when the wrapper starts. If it does not exist, it is created and removed again when the program is finished).
  • 2. If the epitope consensus sequences are to be read from the library, make an internal list of the desired library entries.
  • 3. If the input type is a ZIP file, unzip the file and create one new directory for each of the conatined PDB files. Move each PDB file to its corresponding directory.
  • 4. Do a loop over the structures and/or epitope consensus sequences. For each astructure/epitope consensus sequence pair, DSSP and the core program is called with the required parameters. If the input type is a ZIP file, the outputs are put in the appropriate directories.
  • 5. If the epitope library is used, a sum file containing the total number of epitopes each residue is a member of. (Such a file is generated by the core program for each epitope consensus sequence—here a sum of these files is calculated). If input type is a ZIP file, a sum file is generated for each structure and put in the appropriate directory.
  • 6. If the epitope library is used, a file containing the total number of epitopes found from each entry in the epitope library. If the input type is a PDB file, the file contains only one line (with a number of data corresponding to the library size). If the input type is a ZIP file, there is one line for each structure.
  • 7. Depending on the combination of input type (ZIP or single PDB) and epitope consensus sequence source (typed-in or epitope library), different information is returned to the user: Single PDB+typed in epitope: Graph of numbers of epitopes that each residue is a member of. List of found epitopes.
    • ZIP file+typed in epitope: Graphs (one for each structure) of numbers of epitopes that each residue is a member of. Lists (one for each structure) of found epitopes.
    • Single PDB+epitope library: Graph of numbers of epitopes that each residue is a member of (total for the complete library).
    • ZIP file+epitope library: Graphs (one for each structure) of numbers of epitopes that each residue is a member of (total for the complete library).
    • Data flow sheets for the four different are shown in the FIG.
    • 8. For all modes except Single PDB +typed in epitope, a ZIP file containing all output files is created and returned to the user.
      Immunisation of Brown Norway Rats

Twenty intratracheal (IT) immunisations were performed weekly with 0.100 ml 0.9% (wt/vol) NaCl (control group), or 0.100 ml of a protein dilution (˜0.1−1 mg/ml). Each group contained 10 rats. Blood samples (2 ml) were collected from the eye one week after every second immunisation. Serum was obtained by blood clothing and centrifugation and analysed as indicated below.

Immunisation of Balb/C Mice

Twenty subcutaneous (SC) immunisations were performed weekly with 0.05 ml 0.9% (wt/vol) NaCl (control group), or 0,050 ml of a protein dilution (˜0.01−0.1 mg/ml). Each group contained 10 female Balb/C mice (about 20 grams) purchased from Bomholdtgaard, Ry, Denmark. Blood samples (0.100 ml) were collected from the eye one week after every second immunisation. Serum was obtained by blood clothing and centrifugation and analysed as indicated below.

ELISA Procedure for Detecting Serum Levels of IgE and IqG

Specific IgG1 and IgE levels were determined using the ELISA specific for mouse or rat IgG1 or IgE. Differences between data sets were analysed by using appropriate statistical methods.

Activation of CovaLink Plates

A fresh stock solution of cyanuric chloride in acetone (10 mg/ml) is diluted into PBS, while stirring, to a final concentration of 1 mg/ml and immediately aliquoted into CovaLink NH2 plates (100 microliter per well) and incubated for 5 minutes at room temperature. After three washes with PBS, the plates are dryed at 50° C. for 30 minutes, sealed with sealing tape, and stored in plastic bags at room temperature for up to 3 weeks.

Mouse anti-Rat IgE was diluted 200× in PBS (5 microgram/ml). 100 microliter was added to each well. The plates were coated overnight at 4° C.

Unspecific adsorption was blocked by incubating each well for 1 hour at room temperature with 200 microliter blocking buffer. The plates were washed 3× with 300 microliter washing buffer.

Unknown rat sera and a known rat IgE solution were diluted in dilution buffer: Typically 10×, 20× and 40× for the unknown sera, and ½ dilutions for the standard IgE starting from 1 μg/ml. 100 microliter was added to each well. Incubation was for 1 hour at room temperature.

Unbound material was removed by washing 3× with washing buffer. The anti-rat IgE (biotin) was diluted 2000× in dilution buffer. 100 microliter was added to each well. Incubation was for 1 hour at room temperature. Unbound material was removed by washing 3× with washing buffer.

Streptavidin was diluted 1000× in dilution buffer. 100 microliter was added to each well. Incubation was for 1 hour at room temperature. Unbound material was removed by washing 3× with 300 microliter washing buffer. OPD (0.6 mg/ml) and H₂O₂ (0.4 microliter/ml) were dissolved in citrate buffer. 100 microliter was added to each well. Incubation was for 30 minutes at room temperature. The reaction was stopped by addition of 100 microliter H₂SO₄. The plates were read at 492 nm with 620 nm as reference.

Similar determination of IgG can be performed using anti Rat-lgG and standard rat lgG reagents.

Similar determinations of IgG and IgE in mouse serum can be performed using the corresponding species-specific reagents.

Direct IgE Assay

To determine the IgE binding capacity of protein variants one can use an assay, essentially as described above, but using sequential addition of the follwing reagents:

• • 1) Mouse anti-rat IgE antibodies coated in wells;
  • 2) Known amounts of rat antiserum containing igE against the parent protein;
  • 3) Dilution series of the protein variant in question (or parent protein as positive control);
  • 4) Rabbit anti-parent antibodies
  • 5) HRPO-labelled anti-rabbit Ig antibodies for detection using OPD as described.

The relative IgE binding capacity (end-point and/or affinity) of the protein variants relative to that of the parent protein are determined from the dilution-response curves. The IgE-positive serum can be of other animals (including humans that inadvertently have been senstitized to the parent protein) provided that the species-specific anti-IgE capture antibodies are changed accordingly.

Competitive ELISA (C-ELISA)

C-ELISA was performed according to established procedures. In short, a 96 well ELISA plate was coated with the parent protein. After proper blocking and washing, the coated antigen was incubated with rabbit anti-enzyme polyclonal antiserum in the presence of various amounts of modified protein (the competitior). The residual amount of rabbit antiserum was detected by horseraddish peroxidase-labelled pig anti-rabbit immunoglobulin.

Protein Sequences and Alignments

For purposes of the present invention, the degree of homology may be suitably determined by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45).

Subtilisin Proteases

In the present invention, corresponding (or homologous) positions in subtilisin protease sequences are defined by alignment with Subtilisin Novo (BPN′) from B. amyloliquefaciens, as shown in Table 1A for Alcalase, Protease B, Esperase, Protease C, Protease D, Protease E, Protease A, PD498, Properase, Relase, Savinase. Table 1A: Alignment of different proteases to the sequence of BPN′

Alcalase

69.5% identity in 275 residues overlap; Score: 953.0; Gap frequency: 0.4%

Alcalase,	1	AQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYN-TD	(SEQ ID NO: 45)
BPN′,	1	AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD	(SEQ ID NO: 46)
		** ****   ***     **  * ****** * **  ***** * **** *  *  *  *
Alcalase,	60	GNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMD
BPN′,	61	DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD
		* ************ *  ********  *******   *** ** *  *****  * **
Alcalase,	120	VINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAV
BPN′,	121	VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV
		******** *** * * *** * * ********** ** *   * *** ** ********
Alcalase,	180	DSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPN
BPN′,	181	DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN
		**   ******** ** *****    ** * * *  ***********************
Alcalase,	240	LSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
BPN′,	241	WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ
		***  *  * * ** *********** ****

Protease B

59.6% identity in 275 residues overlap; Score: 820.0; Gap frequency: 2.2%

PROTEASE B,	1	AQTIPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD	(SEQ ID NO: 47)
BPN′,	1	AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD	(SEQ ID NO: 46)
		**  * * *   *** *  * *** ***** * ** * ****   **** ** * *  **
PROTEASE B,	59	GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMH
BPN′,	61	DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD
		* ******** ***************  ********  ***  * *  * ***  * *
PROTEASE B,	119	VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT
BPN′,	121	VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV
		* * *** ** ** *  **  *   ** **** ** *         **  *    ****
PROTEASE B,	175	DQNNNRASFSQYGAGLDIMAPGVNIQSTYPGSTYASDNGTSMATPHVAGAAALVKQKNPS
BPN′,	181	DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN
		*  * *****  *  ** ***** **** **  *   ****** *********   * *
PROTEASE B,	235	WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR
BPN′,	241	WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ
		* * * *  * ** * **    ** ** *  **

Esperase

54.7% identity in 274 residues overlap; Score: 745.0; Gap frequency: 2.2%

Esperase,	1	QTVPWGISFINTQQAHNRGIFGNGARVAVLDTGI-ASHPDLRIAGGASFISSE-PSYHDN	(SEQ ID NO: 48)
BPN′,	2	QSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQDD	(SEQ ID NO: 46)
		* ** * * *     *  *  *    *** * **  *****  *****   ** *   *
Esperase,	59	NGHGTHVAGTIAALNNSIGVLGVAPSADLYAVKVLDRNGSGSLASVAQGIEWAINNNMHI
BPN′,	62	NSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMDV
		* ******** ***************  *******   ***       ****** ***
Esperase,	119	INMSLGSTSGSSTLELAVNRANNAGILLVGAAGNTGRQG----VNYPARYSGVMAVAAVD
BPN′,	122	INMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAVD
		******  ***  *  **  *   *   * **** *  *    * **  *  * ** ***
Esperase,	175	QNGQRASFSTYGPEIEISAPGVNVNSTYTGNRYVSLSGTSMATPHVAGVAALVKSRYPSY
BPN′,	182	SSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNW
		******  ***    ****   **  ** *    ***** ***** ***  *  *
Esperase,	235	TNNQIRQRINQTATYLGSPSLYGNGLVHAGRATQ
BPN′,	242	TNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ
		** * *     * * **    ** **     * *

Protease C

59.6% identity in 275 residues overlap; Score: 825.0; Gap frequency: 2.2%

Protease C,	1	AQSVPWGISRVQAPAAHNRGLTGSGVRVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD	(SEQ ID NO: 49)
BPN′,	1	AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD	(SEQ ID NO: 46)
		***** * *  *** *  * *** * *** * ** * ****   **** ** * *  **
Protease C,	59	GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSYSSIAQGLEWAGNNGMH
BPN′,	61	DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD
		* ******** ***************  ********  *** ** *  * ***  * *
Protease C,	119	VASLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT
BPN′,	121	VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV
		*   *** ** ** *  **  *   ** **** ** *         **  *    ****
Protease C,	175	DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS
BPN′,	181	DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN
		*  * *****  *  **  ****  *** **  *   ****** *********   * *
Protease C,	235	WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAAAR
BPN′,	241	WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ
		* * * *  * ** * **    ** ** *  ***

Protease D

59.3% identity in 275 residues overlap; Score: 815.0; Gap frequency: 2.2%

Protease D,	1	AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD	(SEQ ID NO: 50)
BPN′,	1	AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD	(SEQ ID NO: 46)
		***** * *   *** *  * *** ***** * ** * ****   **** ** * *  **
Protease D,	59	GNGHGTHVAGTIAALDNSIGVLGVAPSAELYAVKVLGASGSGAISSIAQGLEWAGNNGMH
BPN′,	61	DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD
		* ******** *** ***********  ********  ***  * *  * ***  * *
Protease D,	119	VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT
BPN′,	121	VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV
		* * *** ** ** *  **  *   ** **** ** *         **  *    ****
Protease D,	175	DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS
BPN′,	181	DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN
		*  * *****  *  **  ****  *** **  *   ****** *********   * *
Protease D,	235	WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR
BPN′,	241	WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ
		* * * *  * ** * **    ** ** *  **

Protease E

58.2% identity in 275 residues overlap; Score: 800.0; Gap frequency: 2.2%

Protease E,	1	AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD	(SEQ ID NO: 51)
BPN′,	1	AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD	(SEQ ID NO: 46)
		***** * *   *** *  * *** ***** * ** * ****   **** ** * *  **
Protease E,	59	GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGGGAISSIAQGLEWAGNNGMH
BPN′,	61	DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD
		* ******** ***************  ********  * *  * *  * ***  * *
Protease E,	119	VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----DSISYPARYANAMAVGAT
BPN′,	121	VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV
		* * *** ** ** *  **  *   ** **** ** *         **  *    ****
Protease E,	175	DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAVLVKHKNPS
BPN′,	181	DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN
		*  * *****  *  **  ****  *** **  *   ****** ******* *   * *
Protease E,	235	WSNVRIRDHLKKTATSLGSTNLYGSGLVNAEAATR
BPN′,	241	WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ
		* *   *  *  * * **    ** ** *  **

Protease A

58.9% identity in 275 residues overlap; Score: 812.0; Gap frequency: 2.2%

Protease A,	1	AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD	(SEQ ID NO: 52)
BPN′,	1	AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD	(SEQ ID NO: 46)
		***** * *   *** *  * *** ***** * ** * ****   **** ** * *  **
Protease A,	59	GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMH
BPN′,	61	DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD
		* ******** ***************  ********  ***  * *  * ***  * *
Protease A,	119	VANLSLGSPSAGGTLEQAVNSATSRGVLVVAASGNSGA----GSISAPASYANAMAVGAT
BPN′,	121	VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV
		* * *** **    *  **  *   ** **** ** *          *  *    ****
Protease A,	175	DQNNNRASFSQYGPGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS
BPN′,	181	DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN
		*  * *****  ** **  ****  *** **  *   ****** *********   * *
Protease A,	235	WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR
BPN′,	241	WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ
		* * * *  * ** * **    ** ** *  **

PD498

47.7% identity in 266 residues overlap; Score: 487.0; Gap frequency: 4.9%

PD498,	13	YGPQNTSTPAAWDVTRGSSTQTVAVLDSGVDYNHPDLARKVIKGYDFIDRDN-NPMDLNG	(SEQ ID NO: 53)
BPN′,	6	YGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDL--KVAGGASMVPSETPNFQDDNS	(SEQ ID NO: 46)
		**      **        *   *** *** *  ****  **  *         *  * *
PD498,	72	HGTHVAGTVAADTNNGIGVAGMAPDTKILAVRVLDANGSGSLDSIASGIRYAADQGAKVL
BPN′,	64	HGTHVAGTVAA-LNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMDVI
		***********  ** *** * **     ** **   ***    *  **  *      *
PD498,	132	NLSLGCECNSTTLKSAVDYAWNKGAVVVAAAGND----NVSRTFQPASYPNAIAVGAIDS
BPN′,	123	NMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAVDS
		* ***    *  ** *** *   * ********       *    *  **  ***** **
PD498,	188	NDRKASFSNYGTWVDVTAPGVNIASTVPNNGYSYMSGTSMASPHVAGLAALLASQGKN--
BPN′,	183	SNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWT
		****  *   ** **** * ** * * *    *********** ***  *   *
PD498,	246	NVQIRQAIEQTADKISGTGTNFKYGK
BPN′,	243	NTQVRSSLQNTTTKL---GDSFYYGK
		* * *     *  *    *  * ***

Properase

58.9% identity in 275 residues overlap; Score: 813.0; Gap frequency: 2.2%

Properase,	1	AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD	(SEQ ID NO: 54)
BPN′,	1	AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD	(SEQ ID NO: 46)
		***** * *   *** *  * *** ***** * ** * ****   **** ** * *  **
Properase,	59	GNGHGTHVAGTIAALNNSIGVLGVAPNAELYAVKVLGASGGGSNSSIAQGLEWAGNNGMH
BPN′,	61	DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD
		* ******** **************   ********  * *  * *  * ***  * *
Properase,	119	VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT
BPN′,	121	VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV
		* * *** ** ** *  **  *   ** **** ** *         **  *    ****
Properase,	175	DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS
BPN′,	181	DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN
		*  * *****  *  **  ****  *** **  *   ****** *********   * *
Properase,	235	WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR
BPN′,	241	WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ
		* * * *  * ** * **    ** ** *  **

Relase

60.7% identity in 275 residues overlap; Score: 858.0; Gap frequency: 1.8%

Relase,	1	AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGIDSTHPDLNIRGGASFVPGE-PSTQD	(SEQ ID NO: 55)
BPN′,	1	AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD	(SEQ ID NO: 46)
		***** * *   *** *  * *** ***** * **** ****   **** ** * *  **
Relase,	60	GNGHGTHVAGTIAALDNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMD
BPN′,	61	DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD
		* ******** *** ***********  ********  ***  * *  * ***  * **
Relase,	120	VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT
BPN′,	121	VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV
		* * *** ** ** *  **  *   ** **** ** *         **  *    ****
Relase,	176	DQNNNRASFSQYGAELDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVLQKNPS
BPN′,	181	DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN
		*  * *****  * ***  ****  *** **  *   ****** ********* * * *
Relase,	236	WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR
BPN′,	241	WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ
		* * * *  * ** * **    ** ** *  **

Savinase

59.6% identity in 275 residues overlap; Score: 821.0; Gap frequency: 2.2%

Savinase,	1	AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD	(SEQ ID NO: 56)
BPN′,	1	AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD	(SEQ ID NO: 46)
		***** * *   *** *  * *** ***** * ** * ****   **** ** * *  **
Savinase,	59	GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMH
BPN′,	61	DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD
		* ******** ***************  ********  ***  * *  * ***  * *
Savinase,	119	VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT
BPN′,	121	VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV
		* * *** ** ** *  **  *   ** **** ** *         **  *    ****
Savinase,	175	DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS
BPN′,	181	DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN
		*  * *****  *  **  ****  *** **  *   ****** *********   * *
Savinase,	235	WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR
BPN′,	241	WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ
		* * * *  * ** * **    ** ** *  **

To find the homologous positions in subtilisin protease sequences not shown in the alignment of Table 1A, the sequence of interest is aligned to the sequence of BPN′ as shown in Table 1B for YaB protease and Subtilisin sendai. The new sequence is aligned to the BPN′ sequence by using the GAP alignment to the most homologous sequence found by the GAP program. GAP is provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45).

The sequence of the YaB protease is disclosed by Kaneko, R.; Koyama, N.; Tsai,Y. -C.; Juang,R. -Y.; Yoda, K.; Yamasaki, M.; Molecular cloning of the structural gene for alkaline elastase YaB, a new subtilisin produced by an alkalophilic Bacillus strain. J. Bacteriol. 171:5232 (1989), it has Swissprot number P20724, and is shown in SEQ ID NO: 35.

The sequence of the Subtilisin sendai is disclosed by Yamagata, Y.; Isshiki, K.; lchishima, E.; Subtilisin Sendai from alkalophilic Bacillus sp.: molecular and enzymatic properties of the enzyme and molecular cloning and characterization of the gene, aprS. Enzyme Microb. Technol. 17:653 (1995), it has SPTREMBL accession number Q45522, and is shown in SEQ ID NO: 34.

• • Identity to savinase: 81,7%
  • identity to savinase: 82,09%

Swissprot: P20724

TABLE 1B
Alignment of YAB protease to BPN′: 55.3% identity
CLUSTAL W (1.7) multiple sequence alignment

YAB	-QTVPWGINRVQAPIAQSRGFTGTGVRVAVLDTGISN-HADLRIRGGASFVPGE-PNISD	(SEQ ID NO: 57)
BPN′	AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD	(SEQ ID NO: 46)
	*:**:*:.:::**  :*:*:**:.*:***:*:**.. *.**:: ****:**.* **:.*
YAB	GNGHGTQVAGTIAALNNSIGVLGVAPNVDLYGVKVLGASGSGSISGIAQGLQWAANNGMH
BPN′	DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD
	.*.***:****:**************.  **.***** :***. * * :*::**  *.*.
YAB	IANMSLGSSAGSATMEQAVNQATASGVLVVAASGNSG----AGNVGFPARYANAMAVGAT
BPN′	VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV
	: *****..:***::: **::*.****:****:**.*    :..**:*.:*...:****.
YAB	DQNNNRATFSQYGAGLDIVAPGVGVQSTVPGNGYASFNGTSMATPHVAGVAALVKQKNPS
BPN′	DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN
	*..*:**:**. *. **::****.:***:*** *.::******:*****.***: .*:*.
YAB	WSNVQIRNHLKNTATNLGNTTQFGSGLVNAEAATR
BPN′	WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ
	*:*.*:*. *:**:*:**::  :*.**:*.:**::

Alignment of Subtilisin sendai to BPN′: 55.6% identity.
CLUSTAL W (1.7) multiple sequence alignment

sendai	NQVTPWGITRVQAPTAWTRGYTGTGVRVAVLDTGIS-THPDLNIRGGVSFVPGE-PSYQD	(SEQ ID NO: 58)
BPN′	AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD	(SEQ ID NO: 46)
	* .*:*:::::**:  ::****:.*:***:*:**. :****:: **.*:**.* *.:**
sendai	GNGHGTHVAGTIAALNNSIGVVGVAPNAELYAVKVLGANGSGSVSSIAQGLQWTAQNNIH
BPN′	DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD
	.*.********:*********:****.: ********  ***. * * :*::*:  **:.
sendai	VANLSLGSPVGSQTLELAVNQATNAGVLVVAATGNNG----SGTVSYPARYANALAVGAT
BPN′	VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV
	* *:***.* ** :*: **::*. :**:****:**:*    *.**.**.:*...:****.
sendai	DQNNNRASFSQYGTGLNIVAPGVGIQSTYPGNRYASLSGTSMATPHVAGVAALVKQKNPS
BPN′	DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN
	*..*:*****. *. *:::****.**** ***:*.: .*****:*****.***: .*:*.
sendai	WSNTQIRQHLTSTATSLGNSNQFGSGLVNAEAATR
BPN′	WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ
	*:***:*. * .*:*.**:*  :*.**:*.:**::

These alignements reveal that that homology between various subtilisin proteases ranges between 100% and 40%.

Unless specified, subtilisin sequences and positions mentioned in the present invention, are given in the BPN′ numeration, and can be converted by alignement as described above (Tables 1A and 1B).

Sequence identities between different pairs of proteases are given below: Sequence identity to BPN′:

	Savinase	60.4%
	Alcalase	69.5%
	BLAPR	60.4%
	ProteaseC	0.4%
	ProteaseD	0.0%
	ProteaseE	8.2%
	Protease A	0.0%
	Properase	9.6%
	Relase	61.5%
	PD498	44.8%
	sendai	55.6%
	YAB	55.3%

Sequence Identity to Savinase

	Alcalase	60.9%
	BLAPR	98.1%
	ProteaseC	8.5%
	ProteaseD	8.9%
	ProteaseE	6.7%
	Protease A	7.8%
	Properase	8.9%
	Relase	98.1%
	PD498	44.3%
	sendai	81.4%
	YAB	81.8%

Structures

The protein structure of PD498 is disclosed in WO 98/35026 (Novo Nordisk). The structure of Savinase can be found in BETZEL et al, J. MOL. BIOL., Vol. 223, p. 427, 1992 (1svn.pdb).

Homology Modelling

Three dimensional structural models of the subtilisins properase, relase, ProteaseC, ProteaseD, ProteaseE, and PROTEASE B were constructed based on three dimensional structure of Savinase (Protein Data Bank entry 1SVN; Betzel, C., Klupsch, S., Papendorf, G., Hastrup, S., Branner, S., Wilson, K. S.: Crystal structure of the alkaline proteinase Savinase from Bacillus lentus at 1.4 A resolution. J Mol Biol 223 pp. 427 (1992)) using the Modeller 5o ({overscore (S)} ali, A.; T. L. Blundell, “Definition of general topological equivalence in protein structures: A procedure involving comparison of properties and relationships through simulated annealing and dynamic programming,” J. Mol. Biol., 212 403-428 (1990)) module of the Insight 2000 molecular modelling package (Biosym inc.). Default parameters were used with the alignments shown in FIG. 1A as input, e.g. alignment between the columns labelled Savinase and PROTEASE B served as input alignment in construction of a PROTEASE B structural model. The Modeller module by default output ten structural models, of these the model with lowest ‘modeller objective function’ score was chosen as representing PROTEASE B structure.

Lipase

The sequence of the T. lanuginosus lipase (trade name Lipolase) is provided in SEQ ID NO: 1 and the structure is disclosed in WO 98/35026 and as “1tib”, available in Structural Classification of Proteins (SCOP) on the Internet.

Amylase

The amylase used in the examples is the alpha-amylase of Bacillus halmapalus (WO 96/23873), which is called amylase SP722 (the wild-type). Its sequence is shown in SEQ ID NO: 2 and the corresponding protein structure was built from the BA2 structure, as described in WO 96/23874. The first four amino acids of the structural model are not defined, hence the sequence used for numeration of amino acid residues in the examples of this invention is four amino acids shorter than the one of the full length protein SP722.

Several variants of this amylase are available (WO 96/23873). One particularly useful variant has deleted two amino acid residues at D-G at positions 183 and 184 of the SEQ ID NO: 2 (corresponding to residues 179 and 180 of the modelled structure). This variant is called JE-1 or Natalase.

Another amylase that is particularly useful is the amylase AA560: This alkaline alpha-amylase may be derived from a strain of Bacillus sp. DSM 12649. The strain was deposited on 25th Jan. 1999 by the assignee under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at Deutshe Sammmlung von Microorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig DE.

Laccase

The laccase used in this invention is that from Coprinus cinereus (WO 98/38287), the sequence of which is shown as SEQ ID NO: 3. The structure of the Myceliophthora thermophila laccase can be built by homology modeling to the Coprinus cinereus laccase as shown in WO 98/38287.

Cellulase

The cellulase sequence and structure used in the present invention is that of the core fragment of endoglucanase V from Humicola insolens (aka Cel45 or Carezyme). The core fragment structure is available as 3eng.pdb (G. J.DAVIES et al. ACTA CRYSTALLOGR.,SECT.D, Vol. 52, p. 7 1996; G. J.DAVIES et al. BIOCHEMISTRY, V. 34, p. 16210, 1995); SwissProt accession number P43316, and the sequences shown in SEQ ID 4. The corresponding full-length sequence is disclosed in WO 91/17243 and shown here in SEQ ID NO: 5. The numeration of all description and claims of this invention pertain to the core fragment, however, it is contemplated that all claims are also valid for the corresponding positions in the full-lengthprotein.

TABLE 1
Alignment and numeration scheme for subtilisins (SEQ ID NOS: 46, 45, 47, 48, 49, 50, 51, 52, 54, 55, 56, 53, respectively)

BPN′

Alcalase

ProteaseB

Esperase

ProteaseC

ProteaseD

ProteaseE

ProteaseA

Properase

Relase

Savinase

PD498

−6

W

−5

S

−4

P

−3

N

−2

D

−1

P

1

A

A

A

A

A

A

A

A

A

A

Y

2

Q

Q

Q

Q

Q

Q

Q

Q

Q

Q

Q

Y

3

S

T

T

T

S

S

S

S

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L

D

L

L

L

L

L

L

L

L

M

218

N

N

N

S

N

N

N

N

N

N

N

S

219

G

G

G

G

G

G

G

G

G

G

G

G

220

T

T

T

T

T

T

T

T

T

T

T

T

221

S

S

S

S

S

S

S

S

S

S

S

S

222

M

M

M

M

M

M

M

M

M

M

M

M

223

A

A

A

A

A

A

A

A

A

A

A

A

224

S

S

T

T

T

T

T

T

T

T

T

S

225

P

P

P

P

P

P

P

P

P

P

P

P

226

H

H

H

H

H

H

H

H

H

H

H

H

227

V

V

V

V

V

V

V

V

V

V

V

V

228

A

A

A

A

A

A

A

A

A

A

A

A

229

G

G

G

G

G

G

G

G

G

G

G

G

230

A

A

A

V

A

A

A

A

A

A

A

L

231

A

A

A

A

A

A

A

A

A

A

A

A

232

A

A

A

A

A

A

V

A

A

A

A

A

233

L

L

L

L

L

L

L

L

L

L

L

L

234

I

I

V

V

V

V

V

V

V

V

V

L

235

L

L

K

K

K

K

K

K

K

L

K

A

236

S

S

Q

S

Q

Q

H

Q

Q

Q

Q

S

237

K

K

K

R

K

K

K

K

K

K

K

Q

238

H

H

N

Y

N

N

N

N

N

N

N

G

239

P

P

P

P

P

P

P

P

P

P

P

K

240

N

N

S

S

S

S

S

S

S

S

S

N

241

W

L

W

Y

W

W

W

W

W

W

W

242

T

S

S

T

S

S

S

S

S

S

S

243

N

A

N

N

N

N

N

N

N

N

N

N

244

T

S

V

N

V

V

V

V

V

V

V

V

245

Q

Q

Q

Q

Q

Q

R

Q

Q

Q

Q

Q

246

V

V

I

I

I

I

I

I

I

I

I

I

247

R

R

R

R

R

R

R

R

R

R

R

R

248

S

N

N

Q

N

N

D

N

N

N

N

Q

249

S

R

H

R

H

H

H

H

H

H

H

A

250

L

L

L

I

L

L

L

L

L

L

L

I

251

Q

S

K

N

K

K

K

K

K

K

K

E

252

N

S

N

Q

N

N

K

N

N

N

N

Q

253

T

T

T

T

T

T

T

T

T

T

T

T

254

T

A

A

A

A

A

A

A

A

A

A

A

255

T

T

T

T

T

T

T

T

T

T

T

D

256

K

Y

S

Y

S

S

S

S

S

S

S

K

257

L

L

L

L

L

L

L

L

L

L

L

I

258

G

G

G

G

G

G

G

G

G

G

G

S

259

D

S

S

S

S

S

S

S

S

S

S

G

260

S

S

T

P

T

T

T

T

T

T

T

T

261

F

F

N

S

N

N

N

N

N

N

N

G

262

Y

Y

L

L

L

L

L

L

L

L

L

T

263

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

N

264

G

G

G

G

G

G

G

G

G

G

G

F

264a

K

265

K

K

S

N

S

S

S

S

S

S

S

Y

266

G

G

G

G

G

G

G

G

G

G

G

G

267

L

L

L

L

L

L

L

L

L

L

L

K

268

I

I

V

V

V

V

V

V

V

V

V

I

269

N

N

N

H

N

N

N

N

N

N

N

N

270

V

V

A

A

A

A

A

A

A

A

A

S

271

Q

E

E

G

E

E

E

E

E

E

E

N

272

A

A

A

R

A

A

A

A

A

A

A

K

273

A

A

A

A

A

A

A

A

A

A

A

A

274

A

A

T

T

A

T

T

T

T

T

T

V

275

Q

Q

R

Q

R

R

R

R

R

R

R

R

276

Y

EXAMPLES Example 1 Identification of Epitope Sequences and Epitope Patterns

High diversity libraries (10¹²) of phages expressing random hexa-, nona- or dodecapetides as part of their membrane proteins, were screened for their capacity to bind purified specific rabbit lgG, and purified rat and mouse IgGl and IgE antibodies. The phage libraries were obtained according to prior art (se WO 9215679 hereby incorporated by reference).

The antibodies were raised in the respective animals by subcutaneous, intradermal, or intratracheal injection of relevant proteins (e.g. proteases, lipolytic enzymes, amylases, oxidoreductases) dissolved in phosphate buffered saline (PBS). The respective antibodies were purified from the serum of immunised animals by affinity chromatography using paramagnetic immunobeads (Dynal AS) loaded with pig anti-rabbit lgG, mouse anti-rat IgG1 or IgE, or rat anti-mouse IgGl or IgE antibodies.

The respective phage libraries were incubated with the IgG, IgG1 and IgE antibody coated beads. Phages, which express oligopeptides with affinity for rabbit IgG, or rat or mouse IgG1 or IgE antibodies, were collected by exposing these paramagnetic beads to a magnetic field. The collected phages were eluted from the immobilised antibodies by mild acid treatment, or by elution with intact enzyme. The isolated phages were amplified as know to the specialist. Alternatively, immobilised phages were directly incubated with E. coli for infection. In short, F-factor positive E. coli (e.g. XL-1 Blue, JM101, TG1) were infected with M13-derived vector in the presence of a helper-phage (e.g. M13K07), and incubated, typically in 2×YT containing glucose or IPTG, and appropriate antibiotics for selection. Finally, cells were removed by centrifugation. This cycle of events was repeated 2-5 times on the respective cell supernatants. After selection round 2, 3, 4, and 5, a fraction of the infected E. coli was incubated on selective 2×YT agar plates, and the specificity of the emerging phages was assessed immunologically. Thus, phages were transferred to a nitrocellulase (NC) membrane. For each plate, 2 NC-replicas were made. One replica was incubated with the selection antibodies, the other replica was incubated with the selection antibodies and the immunogen used to obtain the antibodies as competitor. Those plaques that were absent in the presence of immunogen, were considered specific, and were amplified according to the procedure described above.

The specific phage-clones were isolated from the cell supernatant by centrifugation in the presence of polyethylenglycol. DNA was isolated, the DNA sequence coding for the oligopeptide was amplified by PCR, and the DNA sequence was determined, all according to standard procedures. The amino acid sequence of the corresponding oligopeptide was deduced from the DNA sequence.

Thus, a number of peptide sequences with specificity for the protein specific antibodies, described above, were obtained. These sequences were collected in a database, and analysed by sequence alignment to identify epitope patterns. For this sequence alignment, conservative substitutions (e.g. aspartate for glutamate, lysine for arginine, serine for threonine) were considered as one. This showed that most sequences were specific for the protein the antibodies were raised against. However, several cross-reacting sequences were obtained from phages that went through 2 selection rounds only. In the first round 22 epitope patterns were identified.

In further rounds of phage display, more antibody binding sequences were obtained leading to more epitope patterns. Further, the literature was searched for peptide sequences that have been found to bind environmental allergen-specific antibodies (J All Clin Immunol 93 (1994) pp. 34-43; Int Arch Appl Immunol 103 (1994) pp. 357-364; Clin Exp Allergy 24 (1994) pp. 250-256; Mol Immunol 29 (1992) pp. 1383-1389; J Immunol 121 (1989) pp. 275-280; J. Immunol 147 (1991) pp. 205-211; Mol Immunol 29 (1992) pp. 739-749; Mol Immunol 30 (1993) pp. 1511-1518; Mol Immunol 28 (1991) pp. 1225-1232; J. Immunol 151 (1993) pp. 7206-7213). These antibody binding peptide sequences were included in the database.

A first generation database of antibody binding peptides identified and their corresponding epitope patterns are shown in Table 2-7 below.

Tables 2-7: Overview of the antibody binding peptide sequences, epitope patterns and epitope sequences. The type of antibody used for identifying the antibody binding sequences is indicated as IgG or IgE and the species from which the antibodies were derived are indicated as mo (mouse), ra (rat) and hu (human).

TABLE 2
Savinase antibody binding peptide sequences, epitope
patterns and epitope sequences.

Antibody	Method of
binding	identifi-	Epitope
peptide	cation	pattern	Donor	Acceptor	Epitope Sequence (BPN′)	Epitope #	IgG	IgE
VQVYGDTSA	Phage	Q>Y>D>	Savinase	savinase	Q206 V81 Y214 G80 D41	sav1.1	Ra
(SEQ ID NO: 59)	display				T208
LQCVGS	Protein		a-amylase	savinase	L21 Q236 V26 G25 S24	sav19.1		Hu
(SEQ ID NO: 60)	fragments		inhibitor
KRFANTELA	Phage	R/K RF>N	Savinase	savinase	K251 R247 A174 N173	sav6.1	Ra-	Mo
(SEQ ID NO: 61)	display						Mo
LDQIFFTRW	Phage	D/E QIFFT	Savinase	savinase	L42/L75 D41 Q2 I79	sav5.1	Ra
(SEQ ID NO: 62)	display
FNDAFFVKM	Phage		Savinase	savinase	N185 D181 A187 F189	sav11.0	Ra
(SEQ ID NO: 63)	display				V203
ANIPIWSRSA	Phage	>RSA	Savinase	savinase	R145 S144 A142	sav3.2-lac1.0-	Ra
(SEQ ID NO: 64)	display					lip4.0-pd5.0
ANIPIWSRSA	Phage	>RSA	Savinase	savinase	S188 R186 S190 A179	sav3.1-lac1.0-	Ra
(SEQ ID NO: 64)	display					lip4.0-pd5.0
RQSTDFGTT	Phage	RQ>>D/E	Savinase	savinase	R186 Q191 S156	sav2.2	Ra
(SEQ ID NO: 65)	display				Q191 Y192
VQVYGDTSA	Phage	Q>Y>D>	Savinase	savinase	G193/A194/G195 D197	sav1.2	Ra
(SEQ ID NO: 66)	display				S265
RRFSNATRA	Phage	R/K RF>N	Savinase	savinase	K251 R247 A174 N173	sav6.1	Ra-	Mo
(SEQ ID NO: 67)	display						Mo
CTARLRAGNACG	Phage	AR>A	Savinase	savinase	A172/A169 R170 A194	sav10.4	Ra
(SEQ ID NO: 68)	display				G193 N261
LDQIFFTRW	Phage	D/E QIFFT	Savinase	savinase	D60 Q59 I44/I35	sav5.2	Ra
(SEQ ID NO: 69)	display
LDQIFFTRW	Phage	D/E QIFFT	Savinase	savinase	L42/L75 D41 Q2179	sav5.1	Ra
(SEQ ID NO: 69)	display
EQIFFTSGL	Phage	D/E QIFFT	Savinase	savinase	E112 Q109 I79	sav5.4	Ra
(SEQ ID NO: 70)	display
GRFSNSKFK	Phage	L>GRS	Savinase	savinase	L196 G195 R170 S163	sav9.2-je4.0-	Ra
(SEQ ID NO: 71)	display					lip5.1-5.2
AVLRDC	Protein		a-amylase	savinase	A254 V268 L267 R10 D181	sav18.1-pd18.1-		Hu
(SEQ ID NO: 72)	fragments		inhibitor			18.2
LQCVGS	Protein		a-amylase	savinase	L217 Q206 V81 G80 S3	sav19.2		Hu
(SEQ ID NO: 73)	fragments		inhibitor
LRQCNERCV	Phage	RQ>>D/E	Savinase	savinase	L267 R10 Q12 N269 E271	sav2.1	Ra
(SEQ ID NO: 74)	display				R275
SPVTKRASLKIDSKK	Protein		Der p II	savinase	A88 S87/T22 L233 K235	sav16.0-pd7.0		Hu
(SEQ ID NO: 75)	fragments				I246
RQSTDFGTT	Phage	RQ>>D/E	Savinase	savinase	R247 Q245 S240/S242	sav2.3	Ra
(SEQ ID NO: 76)	display
FCTNNCELS	Phage	N>>EL	Savinase	savinase	T143 N173 N140 E136 L135	sav7.2	Ra
(SEQ ID NO: 77)	display
FCTNNCELS	Phage	N>>EL	Savinase	savinase	N117 N116 E112 L111	sav7.1	Ra
(SEQ ID NO: 77)	display
DFHVKYAAQ	Phage		Savinase	savinase		sav8.0	Ra
(SEQ ID NO: 78)	display
VAQYKALPVVLENA	Protein		Fel d I	savinase	L135 P168 V139 L111 E112	sav12.0-pd8.0		Hu
(SEQ ID NO: 79)	fragments				N116
AAYPDV	Protein	A>>>>YP>	a-amylase	savinase	A215 Y214 P40 D41 V81	sav13.0-pd13.1-		Hu
(SEQ ID NO: 80)	fragments		inhibitor			13.2
EQIFFTSGL	Phage	D/E QIFFT	Savinase	savinase	E271 Q12 I8	sav5.3	Ra
(SEQ ID NO: 81)	display
VDAAF	Protein		Poa p IX	savinase	V203 D181 A179 A187 F189	sav15.0-pd12.0	Hu
(SEQ ID NO: 82)	fragments
AVLRDC	Protein		a-amylase	savinase	A232 V234 L250 R247 D197	sav18.2-pd18.1-		Hu
(SEQ ID NO: 83)	fragments		inhibitor			18.2
RAFRRNANW	Phage	AR>A	Savinase	savinase	A272/A273 R275 R19 N18	sav10.1	Ra
(SEQ ID NO: 84)	display				A15/A16
CTARLRAGNACG	Phage	AR>A	Savinase	savinase	A15/A16 R19 L21 R275	sav10.2	Ra
(SEQ ID NO: 85)	display				A272 A273 N269
TFHDAPALQ	Phage		Savinase	savinase	H39 D41 A74/A73 P86 A88	sav4.0	Ra
(SEQ ID NO: 86)	display				L90
CTARVVALGVCG	Phage	AR>A	Savinase	savinase	R145 V147 V149 A151	sav10.3	Ra
(SEQ ID NO: 87)	display				L124/L126 G127
GRFSNSKFK	Phage	L>GRS	Savinase	savinase	L148 G146 R145	sav9.1-je4.0-	Ra
(SEQ ID NO: 88)	display				S144/S141 N140	lip5.1-5.2
RRFANDHTR	Phage	R/K RF>N	savinase	savinase	K27 R45 N43 D41 H39	sav6.2	Ra
(SEQ ID NO: 89)	display				T38/T213
KRFANTEPA	Phage	R/K RF>N	savinase	savinase	K251 R247 A174 N173	sav6.1	Ra-	Mo
(SEQ ID NO: 90)	display						Mo
YKVSAL	Protein		a-amylase	savinase	Y91 K27 V26 S24 G23 L21	sav14.0-pd14.0		Hu
(SEQ ID NO: 91)	fragments		inhibitor
TGKYVS	Protein		a-amylase	savinase	S24 G25 K27 Y91 V93	sav17.0-pd17.1-		Hu
(SEQ ID NO: 92)	fragments		inhibitor			17.2

TABLE 3
PD498 antibody binding peptide sequences, epitope patterns and epitope sequences.

Epitope pattern	Donor	Acceptor	Epitope Sequence (BPN′)	Epitope #	IgG	IgE
	Fel d I	pd498	V198 A254 Q252 Y276 K239 A235 L233 P86	pd8.0		Hu
A>>>>YP>	a-amylase	pd498	*3aA Y1/Y2 P-4/P-1 D-2 V81	pd13.2		Hu
	inhibitor
	Poa p IX	pd498	S182 Y6 G7 P8 T13 P14 A15 A16	pd11.0	Hu
>KL>>	Poa p IX	pd498	Y171 K136 L135 A108 Y113	pd4.4	Hu
	a-amylase	pd498	Y48/Y37 K46 *44aaV A43 L42	pd14.0		Hu
	inhibitor
	Poa p IX	pd498	V196/V198 D197 A174/A176 A169 F163	pd12.0	Hu
KQS	Poa p IX	pd498	A142 A147 V148 K120 Q27 S24/S25	pd2.3	Hu
KQS	pd498	pd498	R44 K89 Q27 S236 K120 G146	pd2.2	Ra
	Der p II	pd498	*28aV T88 *44a K R44 A43 L42	pd7.0		Hu
>KL>>	pd498	pd498	N56/N55 K46 L91 A29/A119 T28	pd4.3	Ra
>KL>>	pd498	pd498	N240/N243 K239 L233/L234 A16 T21 R22	pd4.1	Ra
>KL>>	Poa p IX	pd498	Y37 K46 L91 A114 Y113	pd4.5	Hu
>KL>>	pd498	pd498	N240/N243 K239 L233/L234 A16 T21 R22	pd4.1	Ra
YI>KL	pd498	pd498	Y113 I111 A108/A138 K136 L135	pd3.1	Ra
KQS	pd498	pd498	A115 K145 N243 N240 K239 Q237 S236	pd2.1	Ra
>RY>K/R	pd498	pd498	R94 R53 Y48 Q117 R112 S109/S137	pd1.5-lac2.0	Ra
	PhI p V	pd498	A169 Q167 F163 T162 S160 G193	pd10.0		Hu
YI>KL	pd498	pd498	Y276 I246 K239 L234 S236	pd3.2	Ra
>KL>>	pd498	pd498	N240/N243 K239 L233/L234 R22 P86	pd4.2	Ra
A>>>>YP>	a-amylase	pd498	*3aA Y2 P14 D18 V19	pd13.1		Hu
	inhibitor
KQS	Poa p IX	pd498	A15 A16 V274 K239 Q237 S236	pd2.4	Hu
	a-amylase	pd498	G146 K145 Y141 V139 S137	pd17.2		Hu
	inhibitor
	a-amylase	pd498	A273 V274 L233 R22 D87	pd18.1		Hu
	inhibitor		N10 S12 A15/A16 R275 A273/A249 R247
AR>A	Par j 1 + Par o 1	pd498	A174 D197 S170	pd9.0	Hu + Ra	Hu
	pd498	pd498	R22 G23 L233 S236	pd6.2	Ra
>RY>K/R	pd498	pd498	R94 R53 Y48 P57 K46 L91	pd1.4-lac2.0	Ra
>RY>K/R	pd498	pd498	R94 R53 Y48 P57 K46 L91	pd1.4-lac2.0	Ra
	a-amylase	pd498	L96 R94 S33 V35 Y37	pd15.0		Hu
	inhibitor
>RY>K/R	pd498	pd498	S109/S137 R112 Y141 N144 K145	pd1.3-lac2.0	Ra
>RY>K/R	pd498	pd498	T162 R161 Y192 N191 K186	pd1.2-lac2.0	Ra
>RY>K/R	pd498	pd498	T133/T134 R112 Y141 N144 K145	pd1.1-lac2.0	Ra
	a-amylase	pd498	A92 *44aaV L42 R44 D75	pd18.2		Hu
	inhibitor
	a-amylase	pd498	S236 G238 K239 Y276 V274 S270	pd17.1		Hu
	inhibitor
	a-amylase	pd498	S12 P14 W17 S-5 W-6	pd16.0		Hu
	inhibitor
>RSA	pd498	pd498	S137 R112 S109 A108	pd5.0-lac1.0-lip4.0-
				sav3.1-3.2	Ra
	pd498	pd498	S215 M217 I205 M222 G219	pd6.1	Ra

TABLE 4
Antibody binding peptide sequences, epitope patterns and epitope
sequences for the T. lanuginosus lipase (Lipolase).

Antibody	Method of
binding	identifi-	Epitope
peptide	cation	pattern	Donor	Acceptor	Epitope Sequence	Epitope #	IgG	IgE
QRPPRYELE	Phage	RPPR	lipolase	lipolase		lip1.0	Ra
(SEQ ID NO: 93)	display
ELEYRPPRQ	Phage	>EY	lipolase	lipolase	L124 E129 Y164	lip2.1	Ra
(SEQ ID NO: 94)	display
HEYDMRVAW	Phage	>EY	lipolase	lipolase	H215 E219 Y220	lip2.2	Ra
(SEQ ID NO: 95)	display
HEYPMDFHL	Phage	>EY	lipolase	lipolase	H215 E219 Y220	lip2.2	Ra
(SEQ ID NO: 96)	display
SEYSMSITP	Phage	>EY	lipolase	lipolase	S217 E219 Y220	lip2.3	Ra
(SEQ ID NO: 97)	display
CVWPAHAPLSCG	Phage	>P>>PAP>S	lipolase	lipolase	P253 P250 A243	lip3.0	Ra
(SEQ ID NO: 98)	display				P208/P207
					S214/S216/S217
CSWPSPAPLSCG	Phage	>P>>PAP>S	lipolase	lipolase	P253 P250 A243	lip3.0	Ra
(SEQ ID NO: 99)	display				P208/P207
					S214/S216/S217
CDFPLHAPLSCG	Phage	>P>>PAP>S	lipoase	lipolase	P253 P250 A243	lip3.0	Ra
(SEQ ID NO: 100)	display				P208/P207
					S214/S216/S217
CLFPSPAPRSCG	Phage	>P>>PAP>S	lipolase	lipolase	P253 P250 A243	lip3.0	Ra
(SEQ ID NO: 101)	display				P208/P207
					S214/S216/S217
CDGPAPAPWSCG	Phage	>P>>PAP>S	lipolase	lipolase	P253 P250 A243	lip3.0	Ra
(SEQ ID NO: 102)	display				P208/P207
					S214/S216/S217
CSFPLPAPRSCG	Phage	>P>>PAP>S	lipolase	lipolase	P253 P250 A243	lip3.0	Ra
(SEQ ID NO: 103)	display				P208/P207
					S214/S216/S217
CVYPSPAPWSCG	Phage	>P>>PAP>S	lipolase	lipolase	P253 P250 A243	lip3.0	Ra
(SEQ ID NO: 104)	display				P208/P207
					S214/S216/S217
PEYTMNALS	Phage	>EY	lipolase	lipolase	P218 E219 Y220	lip2.4	Ra
(SEQ ID NO: 105)	display
CSRSAKGARLCG	Phage	>RSA	lipolase	lipolase	R209 S214 A182	lip4.0-lac1.0-	Ra
(SEQ ID NO: 106)	display					pd5.0-sav3.1-
						3.2
LEYPMSASQ	Phage	>EY	lipolase	lipolase	L124 E129 Y164	lip2.1	Ra
(SEQ ID NO: 107)	display
RKLTLSGRS	Phage	L>GRS	lipolase	lipolase	L67 G65 R81 S83/S85	lip5.1-je4.0-	Ra
(SEQ ID NO: 108)	display					sav9.0
RKLTLSGRS	Phage	L>GRS	lipolase	lipolase	L96/L97 G212 R209/R179	lip5.2-je4.0-	Ra
(SEQ ID NO: 109)	display				S214	sav9.0
SYGAPATPAA	Protein		Poa p IX	lipolase	S170 Y171 G172 A173	lip6.0	Hu
(SEQ ID NO: 110)	fragments				P174 A150 T153
PAAGYTPAAP	Protein		Poa p IX	lipolase	A18/A19/A20 G65 Y53 T123	lip7.0	Hu	Hu
(SEQ ID NO: 111)	fragments
YKLAY	Protein		Poa p IX	lipolase	Y138 K74 L75 A68 Y16	lip8.1	Hu
(SEQ ID NO: 112)	fragments
YKLAY	Protein		Poa p IX	lipolase	Y53 K127 L67 A68 Y16	lip8.2	Hu
(SEQ ID NO: 112)	fragments
KYDDYVATLS	Protein		Poa p IX	lipolase	Y194 D167 D165 Y164	lip9.0	Hu
(SEQ ID NO: 113)	fragments				V132 A131 L52 S54
EVKATPAGEL	Protein		Poa p IX	lipolase	E43 V44 K46 A47 T72	lip10.0	Hu
(SEQ ID NO: 114)	fragments
CGYSNAQGVDYWI	Protein		Der p I	lipolase	Y53 S54 N25/N26	lip15.0	Hu	Hu
(SEQ ID NO: 115)	fragments				A18/A19/A20 Q15 V44
VPGIDPNACHYMKC	Protein		Der p II	lipolase	P256 I255 D254 P253 N200	lip16.0		Hu
(SEQ ID NO: 116)	fragments				H198 Y261
SPVTKRASLKIDSKK	Protein		Der p II	lipolase	R179 A182 S216/S217 I238	lip17.0		Hu
(SEQ ID NO: 117)	fragments				K237 I235 D234 S224 K223
IMSALAMVYLGAK	Protein		Ovalbumin	lipolase	V140 Y138 L69 A49 A47	lip18.0	Hu
(SEQ ID NO: 118)	fragments				K46
ELGVRE	Protein		a-amylase	lipolase	E99 L97 G109/G177 V176	lip11.0		Hu
(SEQ ID NO: 119)	fragments		inhibitor		R175 D242
GCRKEV	Protein		a-amylase	lipolase	G106 C107 R108 K98 E99	lip12.0		Hu
(SEQ ID NO: 120)	fragments		inhibitor
LRSVYQ	Protein		a-amylase	lipolase	L147 R81 S79 V77 Y16 Q15	lip13.0		Hu
(SEQ ID NO: 121)	fragments		inhibitor
SGPWSW	Protein		a-amylase	lipolase	S170 G172 P174 W89 S83	lip14.0		Hu
(SEQ ID NO: 122)	fragments		inhibitor

TABLE 5
Amylase (Natalase) antibody binding peptide sequences,
epitope patterns and epitope sequences.

Antibody	Method of
binding	identifi-	Epitope
peptide	cation	pattern	Donor	Acceptor	Epitope Sequence	Epitope #	IgG	IgE
ARIDPRGPS	Phage	A>IDP R/K	amylase	amylase	A380 K381 I382 D383 P384	je1.1	Ra
(SEQ ID NO: 123)	display				R389
ARIDPRHGS	Phage	A>IDP R/K	amylase	amylase	A380 K381 I382 D383 P384	je1.1	Ra
(SEQ ID NO: 124)	display				R389
CSVAKIDPRTCG	Phage	A>IDP R/K	amylase	amylase	A109 K138 D140 P142	je1.2	Ra
(SEQ ID NO: 125)	display				R144
CSVAKIDPRTCG	Phage	A>IDP R/K	amylase	amylase	A380 K381 I382 D383 P384	je1.1	Ra
(SEQ ID NO: 125)	display				R389
AKIDPKPDT	Phage	A>IDP R/K	amylase	amylase	A109 K138 D140 P142	je1.2	Ra
(SEQ ID NO: 126)	display				R144
AKIDPKPDT	Phage	A>IDP R/K	amylase	amylase	A380 K381 I382 D383 P384	je1.1	Ra
(SEQ ID NO: 126)	display				R389
ARIDPRHGS	Phage	A>IDP R/K	amylase	amylase	A109 K138 D140 P142	je1.2	Ra
(SEQ ID NO: 127)	display				R144
QIYNDTGPT	Phage	Q>Y>D>	amylase	amylase	Q390 L386 Y368/Y367 D366	je2.4	Ra
(SEQ ID NO: 128)	display
QIYNDTGPT	Phage	Q>Y>D>	amylase	amylase	Q170 I173 Y196 D195	je2.3	Ra
(SEQ ID NO: 128)	display
QIYNDTGPT	Phage	Q>Y>D>	amylase	amylase	Q357 I352 Y349 D366	je2.2	Ra
(SEQ ID NO: 128)	display
QIYNDTGPT	Phage	Q>Y>D>	amylase	amylase	Q331 I370 Y368/Y367 D366	je2.1	Ra
(SEQ ID NO: 128)	display
CGSATIDPRQCG	Phage	A>IDP R/K	amylase	amylase	A109 K138 D140 P142	je1.2	Ra
(SEQ ID NO: 129)	display				R144
CNADNQMYPQCG	Phage	A>>>>YP>	amylase	amylase	N29 A27 D26/D25 Y8	je3.1	Ra
(SEQ ID NO: 130)	display				P41/P42
ARIDPRGPS	Phage	A>IDP R/K	amylase	amylase	A109 K138 D140 P142	je1.2	Ra
(SEQ ID NO: 131)	display				R144
CGSATIDPRQCG	Phage	A>IDP R/K	amylase	amylase	A380 K381 I382 D383 P384	je1.1	Ra
(SEQ ID NO: 132)	display				R389
CDADSSGYPLCG	Phage	A>>>>YP>	amylase	amylase	A107/A109 D108 Y57	je3.3	Ra
(SEQ ID NO: 133)	display				P41/42
QLYGDEQLP	Phage	Q>Y>D>	amylase	amylase	Q331 I370 Y368/Y367 D366	je2.1	Ra
(SEQ ID NO: 134)	display
QLYGDEQLP	Phage	Q>Y>D>	amylase	amylase	Q357 I352 Y349 D366	je2.2	Ra
(SEQ ID NO: 134)	display
QLYGDEQLP	Phage	Q>Y>D>	amylase	amylase	Q170 I173 Y196 D195	je2.3	Ra
(SEQ ID NO: 134)	display
QLYGDEQLP	Phage	Q>Y>D>	amylase	amylase	Q390 L386 Y368/Y367 D366	je2.4	Ra
(SEQ ID NO: 134)	display
RYAQIDPRW	Phage	A>IDP R/K	amylase	amylase	A380 K381 I382 D383 P384	je1.1	Ra
(SEQ ID NO: 135)	display				R389
RYAQIDPRW	Phage	A>IDP R/K	amylase	amylase	A109 K138 D140 P142	je1.2	Ra
(SEQ ID NO: 135)	display				R144
GEFNLGRSS	Phage	L>GRS	amylase	amylase	L88 G92 R31 S28	je4.1-sav9.0-	Ra
(SEQ ID NO: 136)	display					lip5.1-5.2
CNADSWGYPRCG	Phage	A>>>>YP>	amylase	amylase	N29 A27 D26/D25 Y8	je3.1	Ra
(SEQ ID NO: 137)	display				P41/P42
CNADNQMYPQCG	Phage	A>>>>YP>	amylase	amylase	N102 A233 D232 Y54	je3.2	Ra
(SEQ ID NO: 138)	display				P41/P42
CNADSWGYPRCG	Phage	A>>>>YP>	amylase	amylase	N102 A233 D232 Y54	je3.2	Ra
(SEQ ID NO: 137)	display				P41/P42
GEFNLGRSS	Phage	L>GRS	amylase	amylase	L62 G63/G76 R78 S79	je4.2-sav9.0-	Ra
(SEQ ID NO: 139)	display					lip5.1-5.2

TABLE 6
Cellulase (Carezyme; Cel45 from Humicola insolens) antibody binding
peptide sequences, epitope patterns and epitope sequences.

Antibody	Method of
binding	identifi-	Epitope
peptide	cation	pattern	Donor	Acceptor	Epitope Sequence	Epitope #	IgG	IgE
CVHAGPRAGTCG	Phage	>G>>AG	carezyme	carezyme	P23 R201 A83 G84	car1.1	Ra
(SEQ ID NO: 140)	display
CVHAGPRAGTCG	Phage	VH>G>	carezyme	carezyme		car2.0	Ra
(SEQ ID NO: 140)	display
CLSGPLAGRVCG	Phage	>G>>AG	carezyme	carezyme	P23 R201 A83 G84	car1.1	Ra
(SEQ ID NO: 141)	display
CRISPWYSVPCG	Phage		carezyme	carezyme		car3.0	Ra
(SEQ ID NO: 142)	display
CLSGPAAGQSCG	Phage	>G>>AG	carezyme	carezyme	P23 R201 A83 G84	car1.1	Ra
(SEQ ID NO: 143)	display
	Phage	A>IDP R/K	je-1	carezyme	R146 I131 D133 P137	car11.2	Ra
	display
CITRGTRAGWCG	Phage	>G>>AG	carezyme	carezyme	P23 R201 A83 G84	car1.1	Ra
(SEQ ID NO: 144)	display
	Phage	AR>A	savinase	carezyme	A191 R200 R201 A83 N81	car6.2	Ra
	display
CLSGPAAGQSCG	Phage	>G>>AG	carezyme	carezyme	T95/S96 G27 P98 A100 G101	car1.2	Ra
(SEQ ID NO: 143)	display
	Phage	A>IDP R/K	je-1	carezyme	A195 R37 I38 D40 L44	car11.1	Ra
	display
	Phage	Q>Y>D>	savinase,	carezyme	Q59 Y54 G134 D133 T136	car10.0	Ra
	display	>P>>AP>S	je-1		W62/W169 P61 P165 A162
	Phage		lipoprime	carezyme	P160	car9.0	Ra
	display
CITRGTRAGWCG	Phage	>G>>AG	carezyme	carezyme	T95/S96 G27 P98 A100 G101	car1.2	Ra
(SEQ ID NO: 144)	display
	Phage	R/K RF>N	savinase	carezyme	R7 R170 F174 A177	car7.0	Ra
	display
CLSGPLAGRVCG	Phage	>G>>AG	carezyme	carezyme	T95/S96 G27 P98 A100 G101	car1.2	Ra
(SEQ ID NO: 145)	display
	Phage	AR>A	savinase	carezyme	A1 R4 R7 A177 N176	car6.1	Ra
	display
	Phage	>P>RDTG	laccase	carezyme	D178 P180 R4 D2 S183	car5.0	Ra
	display				R170 R153 Y168 P165 K164
	Phage	>RY>K/R	pd498	carezyme	L163	car4.0	Ra
	display
	Phage	D/E QIFFT	savinase	carezyme	Q36 I38 F41 F29 T197	car8.0	Ra
	display
CLTAGPSAGYCG	Phage	>G>>AG	carezyme	carezyme	T95/S96 G27 P98 A100 G101	car1.2	Ra
(SEQ ID NO: 146)	display
CYTTGRLAGLCG	Phage	>G>>AG	carezyme	carezyme	P23 R201 A83 G84	car1.1	Ra
(SEQ ID NO: 147)	display
CYTTGRLAGLCG	Phage	>G>>AG	carezyme	carezyme	T95/S96 G27 P98 A100 G101	car1.2	Ra
(SEQ ID NO: 147)	display
CVHSGPRAGYCG	Phage	>G>>AG	carezyme	carezyme	P23 R201 A83 G84	car1.1	Ra
(SEQ ID NO: 148)	display
CVHSGPRAGYCG	Phage	VH>G>	carezyme	carezyme		car2.0	Ra
(SEQ ID NO: 148)	display
CVHAGPRAGTCG	Phage	>G>>AG	carezyme	carezyme	T95/S96 G27 P98 A100 G101	car1.2	Ra
(SEQ ID NO: 149)	display
CVHSGPRAGYCG	Phage	>G>>AG	carezyme	carezyme	T95/S96 G27 P98 A100 G101	car1.2	Ra
(SEQ ID NO: 148)	display
CVHSGLSRRLLR	Phage	VH>G>	carezyme	carezyme		car2.0	Ra
(SEQ ID NO: 150)	display
CVTRGPNAGSCG	Phage	>G>>AG	carezyme	carezyme	T95/S96 G27 P98 A100 G101	car1.2	Ra
(SEQ ID NO: 151)	display
CLTAGPSAGYCG	Phage	>G>>AG	carezyme	carezyme	P23 R201 A83 G84	car1.1	Ra
(SEQ ID NO: 152)	display
CVTRGPNAGSCG	Phage	>G>>AG	carezyme	carezyme	P23 R201 A83 G84	car1.1	Ra
(SEQ ID NO: 151)	display
CITSGPRAGNCG	Phage	>G>>AG	carezyme	carezyme	T95/S96 G27 P98 A100 G101	car1.2	Ra
(SEQ ID NO: 153)	display
CITSGPRAGNCG	Phage	>G>>AG	carezyme	carezyme	P23 R201 A83 G84	car1.1	Ra
(SEQ ID NO: 153)	display

TABLE 7
Laccase (Myceliophthora thermopila laccase) antibody binding
peptide sequences, epitope patterns and epitope sequences.

Antibody	Method of
binding	identifi-	Epitope
peptide	cation	pattern	Donor	Acceptor	Epitope Sequence	Epitope #	IgG	IgE
PQSD5PGESQ	Phage	P>S/T DPG	laccase	laccase	P180 R175 T168 D166 P165 G265	lac3.2	Ra
(SEQ ID NO: 154)	display
WPKSDAGDS	Phage	P>>DAG	laccase	laccase	P241 R409 S410/S416 D434 A389	lac4.1	Ra
(SEQ ID NO: 155)	display				G390
PQSDAGVVM	Phage	P>>DAG	laccase	laccase	P241 R409 S410/S416 D434 A389	lac4.1	Ra
(SEQ ID NO: 156)	display				G390
DPVRDTGAG	Phage	>P>RDTG	laccase	laccase	P241 R409 D434 T432 G430/G390	lac5.1	Ra
(SEQ ID NO: 157)	display
GPSRDAGLL	Phage	P>>DAG	laccase	laccase	P241 R409 S410/S416 D434 A389	lac4.1	Ra
(SEQ ID NO: 158)	display				G390
PASDAGRGP	Phage	P>>DAG	laccase	laccase	P241 R409 S410/S416 D434 A389	lac4.1	Ra
(SEQ ID NO: 159)	display				G390
PRDSTGLAL	Phage	P>S/T DPG	laccase	laccase	P378 R379 T442 D443 P445 G446	lac3.1	Ra
(SEQ ID NO: 160)	display
PQSDPGESQ	Phage	P>S/T DPG	laccase	laccase	P378 R379 T442 D443 P445 G446	lac3.1	Ra
(SEQ ID NO: 161)	display
RYPFLRATN	Phage	>RY>K/R	laccase	laccase		lac2.0-	Ra
(SEQ ID NO: 162)	display					pd1.1-1.4
						lac1.0-
						lip4.0-
						pd5.0-
GAARDARSA	Phage	>RSA	laccase	laccase		sav3.1-	Ra
(SEQ ID NO: 163)	display					3.2
PRSDTGFGS	Phage	>P>RDTG	laccase	laccase	P241 R409 D434 T432 G430/G390	lac5.1	Ra
(SEQ ID NO: 164)	display
LPRSDPGGR	Phage	P>S/T DPG	laccase	laccase	P180 R175 T168 D166 P165 G265	lac3.2	Ra
(SEQ ID NO: 165)	display
DPARDTGDV	Phage	>P>RDTG	laccase	laccase	P241 R409 D434 T432 G430/G390	lac5.1	Ra
(SEQ ID NO: 166)	display
APKSDNGIT	Phage	P>>DAG	laccase	laccase	P241 R409 S410/S416 D434 A389	lac4.1	Ra
(SEQ ID NO: 167)	display				G390
PKSDPGTNW	Phage	P>S/T DPG	laccase	laccase	P378 R379 T442 D443 P445 G446	lac3.1	Ra
(SEQ ID NO: 168)	display
PRTDPGWLA	Phage	P>S/T DPG	laccase	laccase	P378 R379 T442 D443 P445 G446	lac3.1	Ra
(SEQ ID NO: 169)	display
LPRSDPGGR	Phage	P>S/T DPG	laccase	laccase	P378 R379 T442 D443 P445 G446	lac3.1	Ra
(SEQ ID NO: 170)	display
PSSDPGARS	Phage	P>S/T DPG	laccase	laccase	P180 R175 T168 D166 P165 G265	lac3.2	Ra
(SEQ ID NO: 171)	display
HVFDKNVTR	Phage		laccase	laccase		lac6.0
(SEQ ID NO: 172)	display
PRSDPGTPT	Phage	P>S/T DPG	laccase	laccase	P378 R379 T442 D443 P445 G446	lac3.1	Ra
(SEQ ID NO: 173)	display
PRSDPGTPT	Phage	P>S/T DPG	laccase	laccase	P180 R175 T168 D166 P165 G265	lac3.2	Ra
(SEQ ID NO: 173)	display
PRDSTGLAL	Phage	P>S/T DPG	laccase	laccase	P180 R175 T168 D166 P165 G265	lac3.2	Ra
(SEQ ID NO: 174)	display
PRTDPGWLA	Phage	P>S/T DPG	laccase	laccase	P180 R175 T168 D166 P165 G265	lac3.2	Ra
(SEQ ID NO: 175)	display
PSSDPGARS	Phage	P>S/T DPG	laccase	laccase	P378 R379 T442 D443 P445 G446	lac3.1	Ra
(SEQ ID NO: 176)	display
PKSDPGTNW	Phage	P>S/T DPG	laccase	laccase	P180 R175 T168 D166 P165 G265	lac3.2	Ra
(SEQ ID NO: 177)	display
WPKSDAGDS	Phage	P>>DAG	laccase	laccase	P350 S349 D80 A79 G78	lac4.2	Ra
(SEQ ID NO: 178)	display
PQSDAGVVM	Phage	P>>DAG	laccase	laccase	P350 S349 D80 A79 G78	lac4.2	Ra
(SEQ ID NO: 179)	display
GPSRDAGLL	Phage	P>>DAG	laccase	laccase	P350 S349 D80 A79 G78	lac4.2	Ra
(SEQ ID NO: 180)	display
PASDAGRGP	Phage	P>>DAG	laccase	laccase	P350 S349 D80 A79 G78	lac4.2	Ra
(SEQ ID NO: 181)	display
APKSDNGIT	Phage	P>>DAG	laccase	laccase	P350 S349 D80 A79 G78	lac4.2	Ra
(SEQ ID NO: 182)	display
WPKSDAGDS	Phage	P>>DAG	laccase	laccase	P300 R234 S211 D213 A296	lac4.3	Ra
(SEQ ID NO: 183)	display
PQSDAGVVM	Phage	P>>DAG	laccase	laccase	P300 R234 S211 D213 A296	lac4.3	Ra
(SEQ ID NO: 184)	display
GPSRDAGLL	Phage	P>>DAG	laccase	laccase	P300 R234 S211 D213 A296	lac4.3	Ra
(SEQ ID NO: 185)	display
PASDAGRGP	Phage	P>>DAG	laccase	laccase	P300 R234 S211 D213 A296	lac4.3	Ra
(SEQ ID NO: 186)	display
APKSDNGIT	Phage	P>>DAG	laccase	laccase	P300 R234 S211 D213 A296	lac4.3	Ra
(SEQ ID NO: 187)	display
DPVRDTGAG	Phage	>P>RDTG	laccase	laccase	P378 R379 D469 T473 G446	lac5.2	Ra
(SEQ ID NO: 188)	display
PRSDTGFGS	Phage	>P>RDTG	laccase	laccase	P378 R379 D469 T473 G446	lac5.2	Ra
(SEQ ID NO: 189)	display
DPARDTGDV	Phage	>P>RDTG	laccase	laccase	P378 R379 D469 T473 G446	lac5.2	Ra
(SEQ ID NO: 190)	display
DPVRDTGAG	Phage	>P>RDTG	laccase	laccase	P60 R59 D51/D53 T10/T12 G30	lac5.3	Ra
(SEQ ID NO: 191)	display
PRSDTGFGS	Phage	>P>RDTG	laccase	laccase	P60 R59 D51/D53 T10/T12 G30	lac5.3	Ra
(SEQ ID NO: 192)	display
DPARDTGDV	Phage	>P>RDTG	laccase	laccase	P60 R59 D51/D53 T10/T12 G30	lac5.3	Ra
(SEQ ID NO: 193)	display
DPVRDTGAG	Phage	>P>RDTG	laccase	laccase	P157/P155 R23 D118 T114 G113	lac5.4	Ra
(SEQ ID NO: 194)	display
PRSDTGFGS	Phage	>P>RDTG	laccase	laccase	P157/P155 R23 D118 T114 G113	lac5.4	Ra
(SEQ ID NO: 195)	display
DPARDTGDV	Phage	>P>RDTG	laccase	laccase	P157/P155 R23 D118 T114 G113	lac5.4	Ra
(SEQ ID NO: 196)	display

Example 2 Localisation of Epitope Sequences and Epitope Areas on the 3D-Structure of Acceptor Proteins

Epitope sequences were assessed manually on the screen on the 3D-structure of the protein of interest, using apropriate software (e.g. SwissProt Pdb Viewer, WebLite Viewer).

In a first step, the identified epitope patterns were fitted with the 3D-structure of the enzymes. A sequence of at least 3 amino acids, defining a specific epitope pattern, was localised on the 3D-structure of the acceptor protein. Conservative mutations (e.g. aspartate for glutamate, lysine for arginine, serine for threonine) were considered as one for those patterns for which phage display had evidenced such exchanges to occur. Among the possible sequences provided by the protein structure, only those were retained where the sequence matched a primary sequence, or where it matched a structural sequence of amino acids, where each amino acid was situated within a distance of 5 Å from the next one. Occasionally, the mobility of the amino acid side chains, as provided by the software programme, had to be taken in to consideration for this criterium to be fulfilled.

Secondly, the remaining anchor amino acids as well as the variable amino acids, i.e. amino acids that were not defining a pattern but were present in the individual sequences identified by phage library screening, were assessed in the area around the various amino acid sequences localised in step 1. Only amino acids situated within a distance of 5 Å from the next one were included.

Finally, an accessibility criterium was introduced. The criterium was that at least half of the anchor amino acids had a surface that was >30% accessible. Typically, 0-2 epitopes were retained for each epitope pattern. In some cases, two different amino acids could with equal probability be part of the epitope (e.g. two leucines located close to each other in the protein 3D-structure). For example, in Savinase two epitopes actually fit to the antibody binding peptide LDQIFFTRW (SEQ ID NO:62): L75 D41 Q2 179 and L42 D41 Q2 179. A shorthand notation for such a situation is: L42/L75 D41 Q2 179.

Thus, a number of epitope sequences were identified and localised on the surface of various proteins. As suggested by sequence alignment of the antibody binding peptides, structural analysis confirmed most of the epitopes to be enzyme specific, with only few exceptions. Overall, most of the identified epitopes were at least partially structural. However, some proteins (e.g. amylase) expressed predominantly primary sequence epitopes. Typically, the epitopes were localised in very discrete areas of the enzymes, and different epitope sequences often shared some amino acids (hot-spots).

The identified epitope sequences are shown in Tables 2-7.

Birch Allergen

Bet v1 (WO 99/47680) was used as the parent protein for identification of epitope sequences that may cross react with enzyme epitopes. The structural coordinates from 1BV1.pdb (Gajhede et al., NAT.STRUCT.BIOL., Vol. 3, p. 1040, 1996) were used as well the corresponding sequence (Swissprot accession number P15494). The epitope pattern P>PAP>S (which had been identified from antibody binding peptides specific for anti-Lipolase antibodies) was found to match three (overlapping) epitope sequences on the surface of Bet v1: Bet v1 1.1: P31 A34 P35 A37 P59 S39/S40; Bet v1 1.2: P63 L62 P59 A37 P35 S39/S40; and Bet v1 1.3: P59 S39/S40 P31 A34 P35 S39/S40.

Example 3 Epitope Areas

It is common knowledge that amino acids that surround binding sequences can affect is binding of a ligand without participating actively in the binding process. Based on this knowledge, areas covered by amino acids with potential steric effects on the epitope-antibody interaction, were defined around the identified epitopes. Practically, all amino acids situated within 5 Å from the amino acids defining the epitope were included. The accessibility criterium was not included for defining epitope areas, as hidden amino acids can have an effect on the surrounding structures.

For Savinase, the following amino acid residues belong to the epitope area that correspond to each epitope sequence indicated in Table 2:

sav1.1	A1	Q2	S3	P5	H39	P40	D41	L42	N43	G63	T66	H67
	A69	G70	T71	A73	A74	L75	N77	S78	I79	G80	V81	L82
	G83	N204	V205	Q206	S207	T208	Y209	P210	S212	T213	Y214	A215
	S216	L217
sav1.2	S153	G154	N155	S156	G157	A158	G160	S161	I162	S163	A169	R170
	A174	M175	A176	V177	G178	R186	F189	S190	Q191	Y192	G193	A194
	G195	L196	D197	I198	V199	T220	R247	K251	A254	T255	S256	T260
	N261	L262	Y263	G264	S265	G266	L267
sav2.1	W6	G7	I8	R10	V11	Q12	A13	P14	A15	A16	R19	L21
	V84	T180	D181	Q182	N183	N184	I198	V199	A200	P201	H226	V227
	A230	L233	V234	K237	N238	H249	L250	T253	A254	T255	S256	L257
	S265	G266	L267	V268	N269	A270	E271	A272	A273	T274	R275
sav2.2	S153	G154	N155	S156	G157	A158	S161	I162	S163	G178	A179	T180
	D181	N184	N185	R186	A187	S188	F189	S190	Q191	Y192	G193	L196
	T220	L262	Y263
sav2.3	A142	T143	G146	V147	L148	Y171	A172	N173	A174	M175	D197	A231
	V234	K235	N238	P239	S240	W241	S242	N243	V244	Q245	I246	R247
	N248	H249	L250	K251
sav3.1	S153	G154	N155	S156	G157	A158	V177	G178	A179	T180	D181	N184
	N185	R186	A187	S188	F189	S190	Q191	Y192	V199	A200	P201	G202
	V203	N218	G219	T220	A223	L262	Y263
sav3.2	L111	E112	G115	N116	M119	A138	V139	N140	S141	A142	S144	R145
	G146	V147	V149	N173	N243
sav4.0	Q2	H17	T22	G23	S24	G25	V26	K27	V28	V30	I35	S37
	T38	H39	P40	D41	L42	N43	I44	R45	G46	T66	A69	G70
	T71	172	A73	A74	L75	N76	N77	I79	G80	V81	L82	G83
	V84	A85	P86	S87	A88	E89	L90	Y91	A92	T208	Y209	P210
	S212	T213	Y214
sav5.1	A1	Q2	S3	V4	I35	S37	H39	P40	D41	L42	N43	I44
	T66	A69	G70	A73	A74	L75	N76	N77	S78	179	G80	V81
	L82	G83	P86	L90	T208	Y214
sav5.2	V30	T33	G34	I35	S37	T38	L42	N43	I44	R45	G46	E54
	S57	T58	Q59	D60	G61	N62	G63	H64	G65	T66	H67	A69
	L90	Y91	A92	K94	P210
sav5.3	V4	P5	W6	G7	I8	S9	R10	V11	Q12	A13	P14	A15
	A16	R19	N269	A270	E271	A272	A273	T274	R275
sav5.4	A1	Q2	P40	D41	F50	L75	N77	S78	I79	G80	V81	V104
	S105	S106	I107	A108	Q109	G110	L111	E112	W113	A114	G115	N116
	Q137	A138	S141	A142	Y214
sav6.1	V139	N140	T143	L148	V149	A151	P168	A169	Y171	A172	N173	A174
	M175	A176	D197	1198	N243	V244	Q245	1246	R247	N248	H249	L250
	K251	N252	T253	A254	S265
sav6.2	Q2	G25	V26	K27	V28	A29	I35	S37	T38	H39	P40	D41
	L42	N43	I44	R45	G46	G47	Q59	T66	A69	G70	A73	A74
	L75	N77	I79	G80	V81	L82	A88	E89	L90	Y91	N117	G118
	M119	H120	V121	S207	T208	Y209	P210	G211	S212	T213	Y214	A215
sav7.1	K27	L31	I107	A108	Q109	G110	L111	E112	W113	A114	G115	N116
	N117	G118	M119	A122	L124	L135	Q137	A138	V139	S141
	A142	R145	V149
sav7.2	V104	I107	A108	L111	S132	A133	T134	L135	E136	Q137	A138	V139
	N140	S141	A142	T143	S144	R145	G146	V147	V149	Y167	P168	Y171
	A172	N173	A174	M175	N243	R247
sav9.1	L111	E112	A114	G115	N116	M119	H120	V121	A122	E136	Q137	A138
	V139	N140	S141	A142	T143	S144	R145	G146	V147	L148	V149	V150
	N173	M175	N243	I246	R247	L250
sav9.2	L126	G127	S128	P129	A152	S153	G154	S161	I162	S163	Y167	P168
	A169	R170	Y171	A172	A176	V177	G178	Q191	Y192	G193	A194	G195
	L196	D197	I198	V199	T260	N261	L262	Y263	G264
sav10.1		Q12	A13	P14	A15	A16	H17	N18	R19	G20	L21	T22
	N76	L82	G83	V84	A85	P86	L233	V234	K237	N238	H249	L250
	T253	N269	A270	E271	A272	A273	T274	R275
sav10.2		V11	Q12	A13	P14	A15	A16	H17	N18	R19	G20	L21
	T22	G23	L233	V234	Q236	K237	N238	H249	L250	T253	A254	T255
	L267	V268	N269	A270	E271	A272	A273	T274	R275
sav10.3		L31	D32	H64	V68	V95	L96	I107	L111	A114	G115	N116
	M119	V121	A122	N123	L124	S125	L126	G127	S128	P129	V139	S141
	A142	T143	S144	R145	G146	V147	L148	V149	V150	A151	A152	S153
	S163	Y167	P168	A169	N173	A174	M175	A176	V177	T220	S221	M222
	T224	P225	V227	A228	A231	N243	I246	R247	L250
sav10.4		P131	S132	A133	L135	E136	V139	A151	A152	S153	G160	S161
	I162	S163	Y167	P168	A169	R170	Y171	A172	N173	A174	A176	Q191
	Y192	G193	A194	G195	L196	R247	S259	T260	N261	L262	Y263	G264
sav11.0		W6	G154	N155	S156	G157	A179	T180	D181	Q182	N183	N184
	N185	R186	A187	S188	F189	S190	Q191	Y192	P201	G202	V203	N204
	V205	L217	N218	G219	T220	L262	Y263
sav12.0		L31	I107	A108	Q109	G110	L111	E112	W113	A114	G115	N116
	N117	G118	A122	L124	S132	A133	T134	L135	Q137	A138	V139	N140
	S141	T143	R145	V149	A151	S163	Y167	P168	A169	R170	Y171	N173
	A174
sav13.0		Q2	S3	P5	T38	H39	P40	D41	L42	N43	H67	G70
	A73	A74	L75	N77	I79	G80	V81	L82	G83	V205	Q206	S207
	T208	Y209	S212	T213	Y214	A215	S216	L217
sav14.0		A16	H17	R19	G20	L21	T22	G23	S24	G25	V26	K27
	V28	A29	V30	I35	I44	R45	G46	G47	V84	A85	P86	S87
	A88	E89	L90	Y91	A92	V93	W113	N117	G118	M119	H120	V121
	A232	L233	K235	Q236	K237	T274
sav15.0		W6	R10	G154	N155	S156	G157	V177	G178	A179	T180	D181
	Q182	N183	N184	N185	R186	A187	S188	F189	S190	Q191	V199	A200
	P201	G202	V203	N218	G219	T220	A223	L257	Y263	L267
sav16.0		A13	A16	H17	G20	L21	T22	G23	S24	G25	V26	V28
	I72	A73	V84	A85	P86	S87	A88	E89	L90	H120	G229	A230
	A231	A232	L233	V234	K235	Q236	K237	N238	P239	S240	W241	I246
	H249	L250	A270	A273	T274
sav17.0		T22	G23	S24	G25	V26	K27	V28	A29	V30	L31	D32
	I35	I44	R45	G46	G47	A48	F50	S87	A88	E89	L90	Y91
	A92	V93	K94	V95	G110	W113	N117	G118	M119	H120	V121	A232
	K235	Q236
sav18.1		W6	G7	I8	S9	R10	V11	Q12	A179	T180	D181	Q182
	N183	N184	N185	R186	A187	I198	V199	A200	P201	V203	H226	V227
	A230	H249	L250	K251	N252	T253	A254	T255	S256	L257	S265	G266
	L267	V268	N269	A270
sav18.2		A13	A16	H17	L21	T22	G23	V26	V28	V84	A85	A88
	V121	L148	Y171	A172	N173	V174	M175	A176	G195	L196	D197	I198
	V199	V227	A228	G229	A230	A231	A232	L233	V234	K235	Q236	K237
	N238	W241	N243	V244	Q245	I246	R247	N248	H249	L250	K251	N252
	T253	A254	Y263	G264	S265	G266	V268	A270	A273	T274
sav19.1		A16	H17	R19	G20	L21	T22	G23	S24	G25	V26	K27
	V28	S87	A88	E89	H120	V121	A232	L233	V234	K235	Q236	K237
	N238	P239	T274
sav19.2		A1	Q2	S3	V4	P5	D41	H64	H67	G70	T71	A74
	L75	N77	S78	I79	G80	V81	L82	G83	G202	V203	N204	V205
	Q206	S207	T208	Y209	Y214	A215	S216	L217	N218	G219	M222

For PD498, the following amino acid residues belong to the epitope area that correspond to each epitope sequence indicated in Table 3:

pd1.1	D105	A108	S109	G110	I111	R112	Y113	A114	A115	D116	Q117	N131
	S132	T133	T134	L135	K136	S137	A138	V139	D140	Y141	A142	W143
	N144	K145	G146	A147
pd1.2	C128	E129	A153	G154	N155	D156	N157	V158	S160	R161	T162	F163
	Q167	S170	G178	A179	I180	D181	D184	R185	K186	A187	S188	F189
	S190	N191	Y192	G193	T194	W195	V196	T220	T262	N263
pd1.3	F50	L104	D105	S106	I107	A108	S109	G110	I111	R112	Y113	A114
	A115	D116	Q117	T133	T134	L135	K136	S137	A138	V139	D140	Y141
	A142	W143	N144	K145	G146	A147
pd1.4	T28	*28aV	A29	V30	D32	S33	G34	V35	Y37	*44aaV		I45
	K46	G47	Y48	D49	F50	I51	R53	D54	N55	N56	P57	M58
	D60	L61	K89	I90	L91	A92	V93	R94	V95	L96	D97	A98
	Y113	A114	Q117	A119
pd1.5	D32	S33	G34	K46	G47	Y48	D49	F50	I51	D52	R53	D54
	N55	P57	M58	L61	L91	A92	V93	R94	V95	L96	D97	A98
	L104	D105	S106	I107	A108	S109	G110	I111	R112	Y113	A114	A115
	D116	Q117	G118	A119	T133	T134	L135	K136	S137	A138	V139	D140
	Y141	A142
pd2.1	V19	T21	I111	R112	Y113	A114	A115	D116	Q117	G118	A119	L122
	D140	Y141	A142	W143	N144	K145	G146	A147	V148	L233	L234	A235
	S236	Q237	G238	K239	N240	N243	V244	Q245	I246	R247	Q248	A249
	A273	V274	R275	Y276
pd2.2	S24	S25	T26	Q27	T28	*28aV	L42	A43	R44	*44aK	*44aaV
	I45	D75	N77	D87	T88	K89	I90	L91	G118	A119	K120	V121
	L122	G146	A147	V148	A232	A235	S236
pd2.3	R22	G23	S24	S25	T26	Q27	T28	*28aV	D87	T88	K89	I111
	A115	G118	A119	K120	V121	L122	S137	A138	V139	D140	Y141	A142
	W143	N144	K145	G146	A147	V148	V149	V150	I175	A231	A232	A235
	S236	N243	I246	R247
pd2.4	W-6	S12	T13	P14	A15	A16	V19	T21	R22	G23	S24	Q27
	L230	A231	L233	L234	A235	S236	Q237	G238	K239	N240	N243	Q245
	I246	S270	N271	K272	A273	V274	R275	Y276
pd3.1	L31	K46	G47	Y48	F50	L91	V93	S103	L104	D105	S106	I107
	A108	S109	G110	I111	R112	Y113	A114	A115	D116	Q117	G118	L122
	L124	C130	S132	T133	T134	L135	K136	S137	A138	V139	D140	Y141
	A142	Q167	P168	Y171	P172
pd3.2	V19	T21	R22	G23	S24	Q27	K120	V121	V148	L230	A231	A232
	L233	L234	A235	S236	Q237	G238	K239	N240	N243	Q245	I246	R247
	Q248	A249	I250	Q252	T253	K272	A273	V274	R275	Y276
pd4.1	W-6	S12	T13	P14	A15	A16	W17	D18	V19	T21	R22	G23
	S24	M84	A85	P86	D87	T88	A142	W143	G146	A147	V148	G229
	L230	A231	A232	L233	L234	A235	S236	Q237	G238	K239	N240	N243
	V244	Q245	I246	R247	Q248	A249	I250	S270	N271	A273	V274	R275
	Y276
pd4.2	W-6	T13	A16	W17	V19	T21	R22	G23	S24	*44aK	A73	A74
	*75aT	G83	M84	A85	P86	D87	T88	A142	G146	G146	A147	V148
	G229	L230	A231	A232	L233	L234	A235	S236	Q237	G232	K239	N240
	N243	V244	Q245	I246	R247	Q248	A249	I250	S270	A273	V274	R275
	Y276
pd4.3	T26	Q27	T28	*28aV	A29	V30	L31	Y37	*44aaV		I45	K46
	G47	Y48	D49	D52	R53	D54	N55	N56	P57	M58	V72	T88
	K89	I90	L91	A92	V93	Y113	A114	A115	Q117	G118	A119	K120
	V121	L122	N123	A147	A228	A232
pd4.4	K46	G47	F50	L91	V93	S103	L104	D105	S106	I107	A108	S109
	G110	I111	R112	Y113	A114	A115	D116	Q117	G118	C130	S132	T133
	T134	L135	K136	S137	A138	V139	D140	Y141	Q167	P168	A169	S170
	Y171	P172	N173	A174
pd4.5	T28	*28aV	A29	V30	L31	V35	D36	Y37	N38	H39	L42	A43
	*44aaV		I45	K46	G47	Y48	F50	N55	N56	P57	M58	K89
	I90	L91	A92	V93	A108	S109	G110	I111	R112	Y113	A114	A115
	D116	Q117	G118	A119	L122
pd5.0	F50	S103	L104	D105	S106	I107	A108	S109	G110	I111	R112	Y113
	A114	A115	D116	Q117	T133	T134	L135	K136	S137	A138	V139	D140
	Y141	A142
pd6.1	Y4	Y6	G7	G63	H64	H67	V68	T71	N155	A179	F189	P201
	G202	V203	N204	I205	A206	S207	V209	G213	Y214	S215	Y216	M217
	S218	G219	T220	S221	M222	A223	S224	P225	H226
pd6.2	W-6	T13	A16	W17	V19	T21	R22	G23	S24	S25	Q27	M84
	A85	P86	D87	T88	G229	L230	A231	A232	L233	L234	A235	S236
	Q237	G238	S270	V274
pd7.0	R22	G23	S24	S25	Q27	T28	*28aV	A29	V30	V35	D36	Y37
	N38	H39	P40	D41	L42	A43	R44	*44aK	*44aaV		T66	A69
	G70	V72	A73	A74	D75	N77	A85	P86	D87	T88	K89	I90
	L91	A119	V121	L122	N123	T208	A228	A231
pd8.0	W-6	T13	A16	W17	T21	R22	G23	Q27	*44aK	A73	A74	*75aT
	G83	M84	A85	P86	D87	T88	K120	V121	I175	A176	V177	G178
	V196	D197	V198	T199	A200	V227	G229	L230	A231	A232	L233	L234
	A235	S236	Q237	G238	K239	N240	N243	Q245	I246	Q248	A249	I250
	Q252	T253	A254	F264	Y265	G266	I268
pd9.0	W-6	Y6	G7	P8	Q9	N10	T11	S12	T13	P14	A15	A16
	W17	D18	V19	T21	M84	V139	W143	V148	V149	A151	P168	A169
	Y171	P172	N173	A174	I175	A176	D181	S182	N183	D184	D197	P201
	L230	L233	L234	K239	N240	N243	V244	Q245	I246	R247	Q248	A249
	I250	E251	Q252	T253	A254	K267	I268	N269	S270	N271	K272	A273
	V274	R275	Y276
pd10.0	L124	L126	G127	C128	E129	C130	N131	L135	V139	A151	A152	A153
	G154	N155	D156	N157	V158	S160	R161	T162	F163	Q167	P168	A169
	S170	Y171	A174	I175	A176	N191	Y192	G193	T194	W195	V196	T262
	N263	F264	*264aK
pd11.0	W-6	S-5	Y2	Y4	Q5	Y6	G7	P8	Q9	N10	T11	S12
	T13	P14	W17	D18	V19	T21	A82	M84	I180	D181	S182	N183
	D184	P201	G202	V203	N204	I205	H226	L233	S270	N271	V274	R275
pd12.0	G127	C128	E129	V139	V148	V149	V150	A151	A152	A153	G154	N155
	D156	V158	R161	T162	F163	Q167	P168	A169	S170	Y171	P172	N173
	A174	I175	A176	V177	G178	N191	Y192	G193	T194	W195	V196	D197
	V198	T199	A200	V227	R247	I250	E251	A254	N263	F264	*264aK
	Y265	G266	I268
pd13.1	W-6	S-5	P-4	D-2	P-1	Y1	Y2	S3	*3aA	Y4	Q5	P8
	Q9	S12	T13	P14	A15	A16	W17	D18	V19	T21	R22	G80
	V81	A82	N271	V274	R275
pd13.2	W-6	S-5	P-4	N-3	D-2	P-1	Y1	Y2	S3	*3aA	Y4	Q5
	P8	Q9	P14	W17	D41	G70	A74	D75	*75aT	N76	N77	G78
	I79	G80	V81	A82	G83	A206	S207	T208	Y214
pd14.0	T28	V35	D36	Y37	N38	H39	P40	D41	L42	A43	R44	*44aK
	*44aaV		I45	K46	G47	Y48	D49	F50	R53	D54	N55	N56
	P57	M58	T66	A69	G70	A73	A74	D75	K89	I90	L91	A92
	V93	R94	Y113	T208
pd15.0	V30	L31	D32	S33	G34	V35	D36	Y37	N38	H39	L42	A43
	*44aaV		K46	Y48	D49	F50	I51	N56	P57	M58	D60	L61
	N62	G63	H64	G65	T66	A69	I90	A92	V93	R94	V95	L96
	D97	A98	G100	S101	G102	S103	S106	I107	G110	S125	L126	V209
	P210	N211	N212
pd16.0	W-6	S-5	P-4	N-3	Y2	G7	P8	Q9	N10	T11	S12	T13
	P14	A15	A16	W17	D18	V19	T21	R22	*75aT	N76	A82	G83
	M84	A85	P86	L233	N269	S270	N271
pd17.1	T11	S12	A15	A16	D18	V19	T21	R22	G23	S24	Q27	L230
	A232	L233	L234	A235	S236	Q237	G238	K239	N240	N243	Q245	I246
	Q248	A249	Q252	T253	N269	S270	N271	K272	A273	V274	R275	Y276
pd17.2	A108	I111	R112	A115	D116	K120	L124	T133	T134	L135	K136	S137
	A138	V139	D140	Y141	A142	W143	N144	K145	G146	A147	V148	V149
	P168	Y171	N173	A174	N243
pd18.1	W-6	T13	A16	W17	V19	T21	R22	G23	S24	S25	*44aK	M84
	A85	P86	D87	T88	K89	G229	L230	A231	A232	L233	L234	A235
	S236	Q237	K239	A249	I250	T253	N269	S270	N271	K272	A273	V274
	R275	Y276
pd18.2	D-2	V30	V35	D36	Y37	N38	H39	P40	D41	L42	A43	R44
	*44aK	*44aaV		I45	K46	G47	Y48	P57	T66	A69	G70	A73
	A74	D75	*75aT	N76	N77	I79	V81	A82	A85	P86	D87	T88
	K89	I90	L91	A92	V93	R94	T208

lip2.1	Y53	F55	V63	L78	F80	W117	V120	A121	D122	T123	L124	R125
	Q126	K127	V128	E129	D130	A131	V132	R133	V140	L159	R160	G161
	N162	G163	Y164	D165	I166	G190
lip2.2	V2	L6	F10	A173	P174	R175	A182	L193	Y194	R195	I196	T197
	P204	R205	Y213	S214	H215	S216	S217	P218	E219	Y220	W221	I222
	I235	V236	K237	I238	E239	I241	D242	A243	G246	N247	N248
lip2.3	V2	L6	F10	A182	L185	T186	L193	Y194	R195	I196	T197	H215
	S216	S217	P218	E219	Y220	W221	I222	I235	V236	K237	I238	E239
	G240	I241	A243	G246	N247	N248
lip2.4	V2	L6	F10	L193	Y194	R195	I196	T197	S216	S217	P218	E219
	Y220	W221	I222	I235	V236	K237	I238	E239	G240	A243	G246	N247
	N248
lip3.0	L93	K94	F95	H110	A173	P174	R175	V176	G177	N178	R179	A182
	L185	T186	L193	R195	N200	D201	I202	P204	R205	L206	P207	P208
	R209	E210	F211	G212	Y213	S214	H215	S216	S217	P218	E219	I238
	E239	G240	I241	D242	A243	T244	G245	N248	?R259?		P250	N251
	I252	P253	D254	I255
lip4.0	R175	V176	G177	N178	R179	A180	F181	A182	E183	F184	L185	T186
	R205	P207	P208	R209	E210	F211	G212	Y213	S214	H215	S216	S217
	I241	D242	N248
lip5.1	A20	Y21	N25	N26	T50	F51	L52	Y53	S54	F55	E56	V63
	T64	G65	F66	L67	A68	L69	I76	V77	L78	S79	F80	R81
	G82	S83	R84	S85	I86	E87	N88	W89	K127	V128	A131	H145
	S146	L147	G148	L151	G266
lip5.2	K94	F95	L96	L97	K98	E99	R108	G109	H110	D111	G112	R175
	V176	G177	N178	R179	A180	F181	A182	E183	F184	R205	P207	P208
	R209	E210	F211	G212	Y213	S214	H215	S216	I241	D242	N248
lip6.0	Q9	F10	N11	F13	A14	S17	V63	F80	R81	W89	L93	F113
	S116	W117	F142	T143	G144	H145	S146	L147	G148	G149	A150	L151
	A152	T153	V154	A155	G156	A157	V168	F169	S170	Y171	G172	A173
	P174	R175	V176	F181	L185	L193	Y194	R195	I196	T197	D201	V203
	P204	L206	P207	H215	H258	Y261	F262	I265
lip7.0	F13	A14	Q15	Y16	S17	A180	A19	A20	Y21	C22	G23	N25
	N26	I34	C36	A40	C41	F51	L52	Y53	S54	F55	E56	V63
	T64	G65	F66	L67	S79	F80	R81	V120	A121	D122	T123	L124
	R125	Q126	K127	V128	L264	I265
lip8.1	L12	F13	A14	Q15	Y16	S17	A18	A19	A20	I34	V44	A49
	T50	F51	L52	F66	L67	A68	L69	D70	N71	T72	N73	K74
	L75	I76	V77	S79	H135	P136	D137	Y138	R139	V140	V141	T143
lip8.2	L12	F13	A14	Q15	Y16	S17	A18	A19	A20	I34	V44	A49
	T50	F51	L52	Y53	S54	F55	G65	F66	L67	A68	L69	D70
	N73	L75	I76	V77	L78	S79	T123	L124	R125	Q126	K127	V128
	E129	D130	A131	T143
lip9.0	L6	F10	N25	N26	D27	A28	A30	G31	T50	F51	L52	Y53
	S54	F55	E56	G65	F66	L67	A68	L69	I76	T123	L124	R125
	Q126	K127	V128	E129	D130	A131	V132	R1333	E134	H135	P136	R139
	V140	V141	F142	G156	L159	R160	G161	N162	G163	Y164	D165	I166
	D167	V168	F169	S170	G190	G191	T192	L193	Y194	R195	I196	Y220
lip10.0	N11	L12	Q15	Y16	I34	T35	C36	C41	P42	E43	V44	E45
	K46	A47	D48	A49	D70	N71	T72	N73	K74
lip11.0	F95	L96	L97	K98	E99	I100	N101	D102	C107	R108	G109	H110
	D111	F113	T114	S115	A150	T153	V154	A173	P174	R175	V176	G177
	N178	R179	F181	V203	P204	R205	L206	P207	P208	R209	F211	G212
	Y213	S214	H215	G240	I241	D242	A243	T244	N248
lip12.0	L96	L97	K98	E99	I100	N101	D102	C104	S105	G106	C107	R108
	G109	H110	T114	S115	V176	G177	N178	A180	F181	F184
lip13.0	N11	L12	F13	A14	Q15	Y16	S17	A182	A19	A20	Y21	N26
	I34	C36	A40	C41	P42	E43	V44	A49	F55	E56	V63	T64
	G65	F66	L67	A68	D70	N73	L75	I76	V77	L78	S79	F80
	R81	G82	S83	R84	W89	W117	L124	V128	V141	F142	T143	G144
	H145	S146	L147	G148	G149	A150	L151	A152	A155
lip14.0	Q9	F10	N11	F13	A14	S17	Y21	R81	G82	S83	R84	S85
	I86	E87	N88	W89	I90	G91	N92	L93	F113	T143	G144	H145
	S146	L147	G149	A150	T153	V168	F169	S170	Y171	A173	P174	R175
	V176	L193	Y194	R195	I196	T197	D201	V203	P204	L206	P207	H215
	H258	Y261	F262	I265	G266
lip15.0	N11	L12	F13	A14	Q15	Y16	S17	A18	A19	A20	Y21	C22
	G23	K24	N25	N26	D27	A28	I34	T35	C36	A40	041	P42
	E43	V44	E45	K46	A47	A49	F51	L52	Y53	S54	F55	E56
	T64	G65	F66	L67	S79	F80	R81	T123	L124	K127	L264	I265
lip16.0	A14	E87	I90	H145	G172	I196	T197	H198	T199	N200	D201	I202
	P204	R205	W221	I222	K223	S224	G225	T226	G246	N247	N254	I252
	P253	D254	I255	P256	A257	H258	L259	W260	Y261	F262	G263	I265
lip17.0	E1	V2	F7	F10	G177	N178	R179	A180	F181	A182	E183	F184
	L185	T186	L193	R195	H198	T199	G212	S214	H215	S216	S217	P218
	E219	Y220	W221	I222	K223	S224	G225	T226	V228	P229	V230	T231
	R232	N233	D234	I235	V236	K237	I238	E239	G240	I241	D242	A243
	T244	G245	G246	I262
lip18.0	Q9	F13	Y16	T32	N33	I34	C41	P42	E43	V44	E45	K46
	A47	D48	A49	T50	F51	L52	L67	A68	L69	D70	N71	T72
	N73	L75	I76	V128	V132	H135	P136	D137	Y138	R139	V140	V141
	F142	Y164	D165	I166	D167	F169	Y194

For Amylase, the following amino acid residues belong to the epitope area that correspond to each epitope sequence indicated in Table 5:

je1.1	N2	G3	T4	R33	P346	Y349	I352	L353	T354	R355	P360	V362
	D366	Y367	M378	K379	A380	K381	I382	D383	P384	I385	L386	E387
	A388	R389	Q390	N391	F392	A393	Y394	I450	T451
je1.2	Y57	D58	Y60	D61	F65	N66	Q67	L104	G105	G106	A107	D108
	A109	T110	E111	A135	W136	T137	K138	F139	D140	F141	P142	G143
	R144	G145	N146	T147	Y148	S149	F151	K152	W153	R154	F158
je2.1	M6	Y8	E10	W11	H12	D26	L30	R33	V325	D326	N327	H328
	D329	S330	Q331	P332	G333	E334	E337	F339	K345	Y349	V362	F363
	Y364	G365	D366	Y367	Y368	G369	I370	P371	T372	H373	S374	V375
	P376	A377	M378	K379	I382	D383	L386
je2.2	L289	L293	V314	P318	T323	F324	V325	D326	F339	K345	P346	L347
	A348	Y349	A350	L351	I352	L353	T354	R355	F356	Q357	G358	Y359
	P360	S361	V362	F363	Y364	G365	D366	Y367	Y368	G369	P376	A377
	M378	K379	I382	I385	R389	Q397
je2.3	N102	V116	E117	V118	P120	R123	D159	G160	V161	D162	W163	Q168
	F169	Q170	N171	R172	I173	Y174	K175	A182	W183	D184	V187	D188
	N193	Y194	D195	Y196	L197	M198	Y199	A200	D201	V202	H236
je2.4	T1	N2	T4	M6	Y8	D26	L30	R31	N32	R33	G34	I35
	V325	D326	F339	K345	Y349	L353	V362	F363	Y364	G365	D366	Y367
	Y368	G369	I370	P376	A377	M378	K379	I382	D383	P384	I385	L386
	E387	A388	R389	Q390	N391	F392	Y394	H417
je3.1	M6	Q7	Y8	F9	E10	L13	H19	W20	N21	R22	L23	R24
	D25	D26	A27	S28	N29	L30	R31	N32	R33	I385	W39	I40
	P41	P42	A43	W44	V52	G53	Y54	Y75	A87	L88	N91	V93
	D98	V100	Y364	Y368
je3.2	Y8	F9	W11	H19	W20	W39	I40	P41	P42	A43	W44	D51
	V52	G53	Y54	G55	A56	Y75	D98	V99	V100	M101	N102	H103
	L104	D195	L197	M198	A200	D201	V202	R230	I231	D232	A233	V234
	K235	H236	I237	E262	H328
je3.3	Y8	F9	H19	W20	W39	I40	P41	P42	A43	W44	K45	G46
	T47	V52	G53	Y54	G55	A56	Y57	D58	L59	Q67	K68	Y75
	D98	V100	L104	G105	G106	A107	D108	A109	T110	E111	A135	W136
	T137	K138	F139	D140	F141	P142
je4.1	L23	D25	D26	A27	S28	N29	L30	R31	N32	R33	G34	I35
	T36	I38	A84	I85	H86	A87	L88	K89	N90	N91	G92	V93
	Q94	V95	Q390
je4.2	A43	W44	K45	L59	Y60	D61	L62	G63	E64	F65	V71	R72
	T73	K74	Y75	G76	T77	R78	S79	Q80	L81	E82	S83	Y148
	W219	Y220	T223	L224

Example 4

Having identified ‘antibody binding peptide’ sequences and by consensus analysis also “epitope patterns” (e.g. >DF>>K>), one can identify potential epitope sequences on the 3-dimensional surface of a parent protein (=acceptor protein) in a semi-automated manner using the following method:

The anchor amino acid residues are transferred to a three dimensional structure of the protein of interest, by colouring D red, F white and K blue. Any surface area having all three residues within a distance of 18 Å, preferably 15 Å, more preferably 12 Å, is then claimed to be an epitope. The relevant distance can easily be measured using e.g. molecular graphics programs like InsightII from Molecular Simulations Inc.

The residues in question should be surface exposed, meaning that the residue should be more than 20% surface exposed, preferably more than 50% surface exposed, more preferably 70% surface exposed. The percentage “surface accessible area” of an amino acid residue of the parent protein is defined as the Connolly surface (ACC value) measured using the DSSP program to the relevant protein part of the structure, divided by the residue total surface area and multiplied by 100. The DSSP program is disclosed in W. Kabsch and C. Sander, BIOPOLYMERS 22 (1983) pp. 2577-2637. The residue total surface areas of the 20 natural amino acids are tabulated in Thomas E. Creighton, PROTEINS; Structure and Molecular Principles, W. H. Freeman and Company, N.Y., ISBN: 0-7167-1566-X (1984).

Substitutions of one or more residue (s) within 18 Å, prefereably 15 Å, more prefereably 12 Å, around the geometrical center of the residues involved in the epitope, for a bigger or smaller residues, may destroy the epitope, and make the protein less antigenic.

Residues involved in epitope is 2, preferably 3 and more prefereably 4

Example 5 Production, Selection, and Evaluation of Enzyme Variants with reduced Antigenicity or Immunogenicity

Epitope sequences and hot-spots amino acids were mutated using standard techniques know to the person skilled in the field (e.g. site-directed mutagenesis, error-prone PCR—see for example Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbour, N.Y.).

In the examples shown below, variants were made by site-directed mutagenesis. Amino acid exchanges giving new epitopes or duplicating existing epitopes, according to the information collected in the epitope-database (See Example 1), were avoided in the mutagenesis process.

Enzyme variants were screened for reduced binding of antibodies raised against the backbone enzyme. Antibody binding was assessed by competitive ELISA as described in the Methods section.

Variants with reduced antibody binding capacity were further evaluated in the mouse SC animal model (See methods section).

The following variants showed reduced IgE and/or reduced IgG levels in the mouse model:

Parent			% IgG	% IgE
protein	Mutations	Target epitope sequences	response	response

Savinase	D181N	Sav11.0; Sav15.0 and Sav18.1.	50	19
		Hot spot amino acid.
Savinase	R170L; Q206E	Sav9.4; Sav10.4; Sav1.1; and	5	34
		Sav19.2
Savinase	R170L, S57P	Sav9.4; Sav10.4	45	12
Savinase	R247E	Sav2.3, Sav6.1, Sav18.2	75	30
		Hot spot amino acid.
Savinase	R247Q	Sav2.3, Sav6.1, Sav18.2	17	20
		Hot spot amino acid.
Savinase	R247H	Sav2.3, Sav6.1, Sav18.2	40	27
		Hot spot amino acid.
Savinase	R247K	Sav2.3, Sav6.1, Sav18.2	74	34
		Hot spot amino acid.

Example 6 Production, Selection, and Evaluation of Enzyme Variants with Reduced Antigenicity or Immunogenicity

Hot-spots or epitopes were mutated using techniques known to the expert in the field (e.g. site-directed mutagenesis, error-prone PCR).

In the examples showed below, variants were made by site-directed mutagenesis. Amino acid exchanges giving new epitopes or duplicating existing epitopes according to the information collected in the epitope-database, were avoided in the mutagenesis process. Enzyme variants were screened for reduced binding of antibodies raised against the backbone enzyme. This antibody binding was assessed by established assays (e.g. competitive ELISA, agglutination assay).

Variants with reduced antibody binding capacity were further evaluated in animal studies.

Mice were immunised subcutanuous weekly, for a period of 20 weeks, with 50 microliters 0.9% (wt/vol) NaCl (control group), or 50 microliters 0.9% (wt/vol) NaCl containing 10 micrograms of protein. Blood samples (100 microliters) were collected from the eye one week after every second immunization. Serum was obtained by blood clothing, and centrifugation.

Specific IgG1 and IgE levels were determined using the ELISA specific for mouse or rat IgG1 or IgE. Differences between data sets were analysed by using appropriate statistical methods.

A. Site-Directed mutagenesis of amino acids defining epitopes, with an effect on IgG1 and/or IgE responses in mice.

Epitope: A 172/A169 R170 A194 G193 N261 Pattern:AR>R>A>N Antibody: IgG1+IgE Backbone: Savinase

The variant carried the mutation R170F.

In a competitive IgE ELISA, this variant was less effective in competing for anti-savinase antibodies, giving a 15% lower endpoint inhibition as compared to the savinase backbone.

Mouse studies revealed an 80% reduction of the specific IgE levels, as compared to savinase backbone (p<0.01). The IgG1 levels were not significantly affected.

Epitope: S216 E219 Y220 Pattern: E Y>M Antibody: IgG1 Backbone: Lipoprime

The variant carried the mutation S216W.

In a competitive IgG ELISA, the variant was less effective in competing for Lipolase antibodies, giving a 38% decrease in endpoint inhibition as compared to the enzyme backbone.

Mouse studies revealed a 69% decrease in specific IgG1 levels, compared to the lipolase backbone (p<0.05). The IgE levels were not significantly affected.

B. Site-directed mutagenesis of epitopes, with examples of epitope duplication, and new epitope formation, respectively, predicted by the epitope-database.

Epitope: T143 N173 N140 E136 L135 Pattern: S/T N N>E L Antibody: IgG1 Backbone: Savinase

The variant carried the mutation E136R.

In a competitive IgG ELISA, the variants was less effective in competing for savinase antibodies, giving a 38% decrease in endpoint inhibition as compared to the savinase backbone.

Mouse studies revealed a dramatic increase in specific IgGl levels, compared to savinase backbone (p<0.01). The IgE levels were not significantly affected.

Mutation E136R establishes an IgG1 epitope of the R Y P R/K pattern, previously identified on PD498. Apparently, this new epitope was more antigenic in mice than the existing epitope. The introduction of a savinase unrelated epitope on the savinase backbone could explain the observed discrepancy between competitive ELISA and animal studies.

In this example, it was found that using information derived exclusively from screening phage libraries with anti-PD498 antibodies (to identify the R Y P R/K epitope pattern of Table 2) one could predict the outcome of a genetic engineering experiment for Savinase in which the E136R mutation created the PD498-epitope on the Savinase surface, leading to increased immunogenicity of this Savinase variant. This demonstrates that the epitope patterns identified may be used to predict the effect on immunogenicity of substitutions in proteins that are different from the parent protein(s) used to identify the epitope pattern.

C. Site-directed mutagenesis of amino acids defining epitope areas, with a differential effect on IqG1 and IgE antibody levels in mice, and an inhibiting effect on IgG binding, respectively.

Epitope: A172/A169 R170 A194 G193 N261 Pattern: A R>R>A>N Antibody: IgG1+IgE Backbone: Savinase Epitope area: P131, S132, A133, L135, E136, V139, A151, A152, S153, G161, S162, I165, S166, Y167, P168, Y171, N173, A174, A176, Q191, Y192, G195, L196, R247, S259, T260, L262, Y263, G264.

The variant was different at position Y167 by the mutation Y1671.

In a competitive IgE ELISA, the variant was less effective in competing for anti-savinase antibodies, giving a 8% lower endpoint inhibition as compared to the its backbone.

Mouse studies revealed an 75% reduction of the specific IgE levels, as compared to the backbone (p<0.01). In contrast, the IgG1 levels were dramatically increased (p<0.01). Epitope: T143 N173 N140 E136 L135 Pattern: S/T N N>E L Antibody: IgG1 Backbone: Savinas Epitope area: V10A, I107, A108, L111, E112, G115, S132, A133, T134, Q137, A138, V139, S141, A142, S144, R145, G146, V147, V149, Y167, P168, Y171, A172, A174, M175, N243, R247.

While variant no. 1 was mutated at the epitope position (N140D), variant no. 2 was mutated at N140 (N140D), but also at the epitope area position (A172D).

In a competitive IgG ELISA, variant no. 1 was less effective in competing for anti-savinase antibodies, as compared to savinase. This variant revealed a 21% lower endpoint inhibition as compared to the its backbone.

Variant no. 2 resulted in an endpoint inhibition that was 60% lower as compared to savinase, and 40% as compared to variant no. 1.

Example 7 Conjugation of Savinase Variant E136K with Activated Bis-PEG-1000

4.9 mg of the Savinase variant was incubated in 50 mM Sodium Borate pH 9.5 with 12 mg of N-succinimidyl carbonate activated bis-PEG 1000 in a reaction volume of approximately 2 ml. The reaction was carried out at ambient temperature using magnetic stirring while keeping the pH within the interval 9.0-9.5 by addition of 0.5 M NaOH. The reaction time was 2 hours.

The derivatives was purified and reagent excess removed by size exclusion chromatography on a Superdex-75 column (Pharmacia) equilibrated in 50 mM Sodium Borate, 5 mM Succinic Acid, 150 mM NaCl, 1 mM CaCl₂ pH 6.0.

The conjugate was stored at −20° C., in the above described buffer.

Compared to the parent enzyme variant, the protease activity of the conjugate was retained (97% using Dimethyl-casein as substrate at pH 9).

Example 8

Competitive ELISA was performed according to established procedures. In short, a 96 well ELISA plate was coated with the parent protein. After proper blocking and washing, the coated antigen was incubated with rabbit anti-enzyme poiyclonal antiserum in the presence of various amounts of modified protein (the competitior).

The amounts of residual rabbit antiserum was detected by pig anti-rabbit immunoglobulin, horseraddish peroxidase labelled.

Epitope: T143 N173 N140 E136 L135 Pattern: S/T N N>E L Antibody: IgG1 Backbone: Savinase Mutation: E136K Modification: bis-NHS-PEG1000

The data show that the derivative (60% endpoint inhibition) has reduced capacity to bind enzyme specific immunoglobulines, as compared to the parent protein (100% endpoint inhibition).

Example 9

For this example the epitope sequences were determined in four environmental allergens (Bet v1; Der f2; Der p2 and PhI p2), based on their structures (1btv.pdb; 1ahm.pdb; a19v.pdb; and 1whp.pdb, respectively), sequences (SEQ ID NOS: 6, 7, 8 and 9, respectively) and computer modelling of the epitope patterns that had been assembled in our database (shown in Table 8). The allergens arise from common sources of allergy: Birch (Bet v1 from Betula pendula), House dust mites (Der f2 from Dermatophagoides farinae and Der p2 from Dermatophagoides pteronyssinus), and Timothy grass (Phl p2 from Phleum pratense).

The protein surface is scanned for epitope patterns matching the given “consensus” sequence of about 6-12 residues. First, residues on the protein surface that match the first residue of the consensus sequence are identified. Within a specified distance from each of these, residues on the protein surface that match the next residue of the consensus sequence are identified. This procedure is repeated for the remaining residues of the consensus sequence. The method is further described under the paragraph “Methods” above and the computer program can be found in the Appendixes.

The critical parameters used in this screening included:

• • i) a maximal distance between the alpha-carbon atoms of subsequent amino acids,
  • ii) a minimal accessability of the amino acid of 20 Å2,
  • iii) the largest maximal distance between the most distinct amino acids should be less than 25 Å,
  • iv) the 5 best epitopes were taken,
  • v) the minimal homology with the epitope pattern of interest was 80%

In this way a number of potential epitopes are identified. The epitopes are sorted according to total surface accessible area, and certain entries removed:

• • 1) Epitopes that contain the same protein surface residue more than once. These are artefacts generated by the described algorithm.
  • 2) Epitopes which are “too big”, i.e. where a distance between any two residues in the epitope exceeds a given threshold.

The epitope sequences found by this second generation mapping procedure were:

The epitope sequences found were:

	Bet v1:
	Epi#02
	A146, K32, Q36, F30, T142, R145, V12
	A34, K32, Q36, F30, T142, R145, V12
	Epi#03
	L62, K65, —, I56, Y66
	L24, K20, H76, I23, Y81
	L24, K20, H76, I104, Y81
	Epi#04
	K134, S136, Q132, K129, A130, A135
	k134, S136, Q132, K129, V128, G1
	Epi#05
	G140, A146, R145, T10, G111, A106, T107, V12
	G26, A146, R145, T10, G110, A106, T107, V12
	G140, A146, R145, T10, G110, S11, S149, L152
	G110, A106, S11, T9, G140, R145, T10, V12
	G140, A146, R145, T10, G111, S11, S149, V12
	Epi#06
	G110, P108, D109, T107, A106, P14
	G111, P108, D109, T107, A106, P14
	A34, N28, D27, S40, K32, P35
	G26, N28, D27, S39, K32, P35
	A106, N78, D75, T77, A16, P14
	G26, N28, D27, S39, Q36, P35
	Epi#07
	G46, T52, D69, S99, R70, V71, P50, D72
	G49, T52, D69, S99, R70, V71, P50, D72
	G48, T52, D69, S99, R70, V71, P50, D72
	Epi#08
	K123, E127, G1, V2, H121, F3
	K65, E60, F64, V67, F58
	K65, E60, F58, V67, F64
	K129, E127, G1, V2, H121, F3
	Epi#09
	S149, L152, D156, N159, R17, L24, D75, K103, N78, A106, V12
	L152, S149, D156, N159, R17, L24, D75, H76, N78, A106, V12
	L152, D156, N159, R17, L24, D75, K80, N78, A106, V12
	Epi#10
	D109, A106, N78, T77, F79, R17, K20
	E141, T10, R145, T142, F30, G26, K32
	E8, T10, R145, T142, F30, G26, K32
	Epi#11
	F30, K32, I38, Q36, V33, E148
	F22, F30, I38, Q36, V33, E148
	F30, L143, I38, Q36, V33, E148
	Epi#12
	Y5, E6
	Y83, E73
	Y120, E127
	Y5, E8
	Y66, E87
	Y81, E73
	Epi#13
	H76, A16, P14, T107, A106, P108, G110, G111
	A16, R17, P14, T107, A106, P108, G110, G111
	A157, R17, P14, T107, A106, P108, G111, G110
	Epi#15
	K65, P90, D93, I91, K97, G92
	K32, P31, D27, I56, K65, G61
	Epi#17
	A153, S149, R145, S11
	A106, S11, R145, S149
	Epi#18
	R145, S149, L152, A153, Y150, L151, H154, S155
	R145, S149, L152, A153, S155, L151, A157, N159
	Epi#22
	D125, D93, P90, K65
	D93, P90, P63, E60
	Epi#23
	K55, N43, E42, S57, L62, P63
	K68, N43, E42, S40, F30, P35
	K54, N43, E42, S57, F64, P63
	K55, N43, E42, S40, F58, P35
	Epi#24
	E96, K97, E87, P90, F64, E60, K65
	E127, K123, E96, P90, F64, P63, K65
	E42, K68, E87, P90, F64, E60, K65
	E42, K55, E87, P90, F64, E60, K65
	D93, G92, E87, P90, F64, E60, K65
	D125, K123, E96, P90, F64, P63, K65
	Epi#25
	R70, K55, I44, E45, E42
	R70, K54, I44, E45, N47
	R70, K68, I53, E45, N47
	Epi#27
	D93, E127, D125, K123
	Epi#28
	A146, Q36, F58, E60, L62, F64, P63, K65
	I38, Q36, F58, E60, L62, F64, P63, K65
	A34, Q36, F58, E60, L62, F64, P63, K65
	L143, Q36, F58, E60, L62, F64, P63, K65
	V33, Q36, F58, E60, L62, F64, G61, K65
	Epi#29
	G61, K65, L62, F58, E60
	I56, K65, L62, F64, E60
	G89, K65, L62, F64, E60
	V67, K65, L62, F64, E60
	Epi#30
	G1, N4, S99, H121, K97, I91, P90
	I113, I13, S149, H154, S155, L152, L151
	I13, L152, A153, H154, S155, L151, V33
	G110, I13, S149, H154, S155, L152, L151
	G1, N4, S99, H121, K97, I98, V2
	G1, N4, S99, H121, K97, I91, V85
	Epi#33
	K32, F30, P35, S39, S57, K65
	Q36, F30, P35, S39, S40, K32
	K32, F30, P35, S40, S57, K65
	K65, F58, P35, S39, A34, R145
	Epi#34
	V105, P14, T107, V12, R145, Y150, S155
	I113, P14, T107, V12, R145, Y150, S155
	Epi#37
	P50, V74, L24, R17, N159
	P50, V74, L24, K20, N159
	P14, R17, L24, K20, N159
	Epi#38
	L143, G140, E141, R145, V33, N28, P31, S39
	L143, G140, E141, R145, V33, N28, P31, S40
	L143, G140, E141, R145, V33, N28, P31, S57
	Epi#39
	A130, E127, H126, T94, P90, G89, L62
	A130, E127, H121, T94, P90, G89, L62
	Epi#40
	A157, L152, A153, Y150, K32, S39
	A153, L152, A157, Y150, K32, S40
	R17, L151, A153, Y150, K32, S40
	R145, L143, A34, Y150, A153, S155
	R145, L143, G140, T9, K115, T10
	Epi#41
	P63, Y66, L62, S57
	Epi#44
	I23, R17, D156, Y150, S149, V12, T10
	L24, R17, D156, Y150, S149, V12, P14
	L24, R17, D156, Y158, A16, A106, P108
	I13, R17, D156, Y150, S149, V12, T10
	L151, R17, D156, Y150, S149, V12, T10
	L24, R17, D156, Y150, S149, V12, T107
	Epi#45
	K32, P35, F30, Y150, R145, M139, G140
	K32, P35, F30, Y150, R145, M139, L143
	K32, P31, F30, Y150, R145, M139, G140
	Epi#47
	L152, S149, R145, L143, A34, F30, N28, P31, P35
	A153, S149, R145, A146, A34, F30, N28, P31, P35
	Epi#48
	E60, K65, P90, P63, G61
	E60, K65, P63, P90, G92
	Epi#51
	T94, H126, E127, D125, G124, K123, H121
	D125, H126, E127, T94, K123, T122, H121
	Der f2:
	Epi#02
	A98, K100, S101, P99, R128, R31
	A98, K100, R128, P99, R31, V94
	T91, N93, P95, P34, R31, R128
	L61, N93, P95, P34, R31, R128
	Epi#03
	L40, K15, A39, I13, Y86
	L40, K14, A39, I88, Y90
	Epi#05
	G32, A98, R31, P34, G20, T36, T91, Y90
	G32, A98, R31, P34, G20, T36, T91, V94
	G32, A98, R31, P34, G20, T36, T91, L37
	G32, A98, R31, P34, G20, T36, T91, V18
	Epi#06
	A98, P99, D129, R31, K96, P95
	G32, P99, D129, R128, R31, P95
	A98, P99, D129, R31, K33, P95
	A98, P99, D129, R31, K96, P34
	A98, P99, D129, R128, K126, P26
	Epi#07
	T107, S57, D59, S101, R128, A98, P99, D129
	T107, S57, D59, S101, R31, A98, P99, D129
	Epi#08
	K15, D87, V76, H74, F75
	K14, D87, V76, H74, F75
	K77, D87, V76, H74, F75
	Epi#09
	L61, D64, I68, H74, F75, T70, N71
	N114, N46, D113, K48, N71, T70, T49
	G83, N46, D113, K48, N71, T70, T49
	Epi#10
	L40, I13, D42, N44, V81, K48, N46, N114, G115
	L40, I13, D42, N44, V81, K82, N46, N114, G115
	L37, D19, G20, V18, V3, D4, K6, A120, T107, V105
	Epi#11
	F75, K51, I111, Q45, V116, D113
	F75, K51, I111, Q45, V81, D113
	Epi#12
	Y90, E38
	Epi#13
	H30, R31, P95, A98, P99, S101, G60, L61
	Epi#15
	K96, P99, D129, I28, R128, A98
	K96, P99, D129, I127, R128, A98
	K96, P99, D129, I29, R128, A98
	K55, P66, D64, I68, T70, G67
	Epi#18
	R31, R128, I28, G125, T123, H124, V105
	R31, R128, I127, G125, T123, H124, V105
	Epi#22
	D1, M17, D4, V3, K6
	D1, M17, D19, P34, K96
	D1, M17, D4, V5, K6
	Epi#23
	K14, N11, E12, N44, Q85, P79
	k14, N11, E12, N10, Q45, P79
	K14, N11, E12, N44, Q84, P79
	K14, N11, E12, L40, Q85, P79
	Epi#24
	D129, K100, E102, P99, R128, R31, K96
	E62, G60, E102, P99, R128, R31, K96
	D129, K126, E102, P99, R128, R31, K33
	D129, K126, E102, P99, R31, P95, K96
	Epi#25
	R31, K96, I97, D59, E62
	R128, R31, I97, D59, E102
	R128, K126, I127, E102, N103
	Epi#27
	D64, E62, D59, K100
	D59, E62, D64, K55
	D87, E38, D19, K33
	D19, E38, D87, K15
	D19, E38, D87, K14
	D19, E38, D87, K77
	Epi#28
	V16, D87, Q85, K14, E12, K15, Q2, D1
	I13, D87, Q85, K14, E12, K15, Q2, D1
	V3, D1, Q2, K15, E12, K14, Q85, D87
	L40, D87, Q85, K14, E12, K15, Q2, D1
	I88, D87, Q85, K14, E12, K15, Q2, D1
	V76, D87, Q85, K14, E12, K15, Q2, D1
	V18, D1, Q2, K15, E12, K14, Q85, D87
	Epi#29
	G32, N93, L61, E62
	V94, N93, L61, E62
	Epi#30
	G60, I97, A98, H30, K96, P34, P95
	I68, N71, H74, K77, P79, V81
	G32, I97, A98, H30, K96, P95, P34
	Epi#34
	V105, P26, S24, G125, R128, S101, P99
	W92, P34, T91, V94, R31, S101, P99
	I28, P26, T123, G125, R128, S101, P99
	Epi#37
	A120, V16, L40, K14, N11
	A39, V16, L40, K14, N11
	Y90, A39, L40, K14, N11
	Y86, A39, L40, K14, N11
	Epi#39
	A120, E38, T91, P34, G20, L37
	A39, E38, T91, P34, G20, L37
	Epi#40
	G20, L37, A120, T123,K6, S24
	A39, L37, A120, T123, K6, S24
	G20, L37, A120, T107, K6, T123
	Epi#41
	P34, L37, V106, S57
	Epi#42
	P26, S24, G125, R128, R31
	P99, S101, G125, R128, R31
	Epi#44
	V16, Q2, D19, P34, W92, Y90, A39, V18, T91
	V16, Q2, D19, P34, W92, Y90, A39, V5, T123
	V3, Q2, D19, P34, W92, Y90, A39, V18, T91
	Epi#45
	K77, H74, F75, N71, D69, G67
	K77, H74, F75, N71, D69, V76
	K77, H74, F75, N71, D69, V65
	Epi#46
	A98, R128, R31, P95, N93, G32
	A98, R128, R31, P34, G20, Q2
	Epi#48
	Q2, D19, P34, P95, G32
	H30, K96, P95, P34, G20
	Epi#49
	D87, D42, L40, Q85, Q84, C78, T47, Q45, K48
	D87, D42, L40, Q85, Q84, C78, T47, Q45, K82
	Epi#50
	D19, W92, P34, T91
	D19, W92, P34, P95
	D19, W92, T91, T36
	Epi#51
	D129, H30, K33, R31, R128, K126, H124
	R31, H30, D129, R128, K100, K126, H124
	T123, H124, K126, R128, R31, K33, H30
	Der p2:
	Epi#03
	L17, K89, A39, I13, Y86
	L17, K89, A72, I88, Y90
	L17, K89, A72, I52, Y90
	Epi#04
	K15, S1, Q2, K14, V16, L17
	K15, S1, Q2, K14, A39, L17
	K15, S1, Q2, K14, V40, I13
	Epi#05
	G60, A56, L61, P99, G32, R31, H30, I97
	G60, A56, L61, P99, G32, R31, H30, I28
	Epi#06
	G60, A56, D64, S57, K55, P66
	G83, N46, D114, T49, K48, P79
	G60, N103, D59, S101, R31, P95
	Epi#08
	K55, D64, S57, V106, F35
	K55, E62, S57, V106, F35
	Epi#09
	L61, G60, E102, R128, I28, K126, N103, T123, V105
	L61, G60, E102, R128, I127, K100, N103, T123, V105
	L61, G60, E102, R128, I127, H124, N103, T123, V105
	Epi#10
	SAS: 435, Size 24.47: D69, T91, N93, F35, G32, R31
	SAS: 422, Size 20.74: E38, T91, N93, F35, G32, K96
	Epi#11
	K14, I13, Q85, V81, E42
	K15, I13, Q85, V81, E42
	K14, I13, Q85, V40, D87
	Epi#12
	Y86, E42
	Y90, E53
	Y90, E38
	Epi#13
	H30, A125, P26, T123, A122, P19, L37, P34, W92
	H30, A125, P26, T123, A122, H124, S24, G23, G20
	H30, A125, P26, T123, A122, P19, L17, G20, F35
	Epi#15
	K55, P66, D69, I68, K89, A72
	K55, P66, D69, I68, K89, A39
	K55, P66, D64, I54, K109, G115
	K55, P66, D64, I54, K109, A9
	Epi#18
	R31, I29, A125, S101, E102, N103
	R31, I29, A125, S101, E102, V104
	R31, I29, A125, T123, A122, V105
	Epi#22
	D69, P66, D64, V65, K55
	D64, P66, D69, T91, K89
	D59, L61, D64, P66, W92
	D59, L61, D64, V65, E62
	D69, P66, D64, V65, E53
	Epi#24
	D64, K55, E62, P99, R31, P34, K96
	E53, K55, E62, P99, R31, P95, K96
	D64, K55, E62, P99, R31, A98, K96
	Epi#25
	R31, H30, I28, E102, N103
	R128, K126, I127, E102, N103
	R128, K126, I28, E102, V105
	Epi#27
	D64, E53, D69, K89
	D69, E53, D64, K55
	D59, E62, D64, K55
	Epi#28
	V40, D87, Q85, E42, Q84, G83, K82
	G20, H22, Q2, L17, E38, L37, Q36, P34, K33
	G20, H22, Q2, L17, E38, L37, F35, P34, K33
	Epi#29
	I97, K100, L61, E62
	G60, N103, L61, E62
	I127, N103, L61, E62
	Epi#30
	G60, N103, S101, H30, K96, I97, P95
	G60, N103, A125, H30, K96, I97, P95
	I28, I127, A125, H30, K96, I97, P95
	Epi#33
	Q36, F35, V106, S57, A56, K55
	K33, F35, V106, S57, A56, K55
	Epi#34
	I28, P26, S24, G23, G20, T123, S57
	I28, P26, S24, V3, G20, T123, T107
	W92, P34, T91, V18, G20, T123, P26
	Epi#37
	P66, V63, L61, K100, N103
	P95, A98, L61, K100, N103
	P19, V18, L17, K89, D87
	P19, V3, L17, K89, D87
	T123, V104, L61, K100, N103
	Epi#38
	L61, G60, E102, A125, V105, N103, P99, S57
	L61, G60, E62, A56, V105, N103, P99, S57
	Epi#39
	A125, E102, H124, T123, P26, G20, L17
	Epi#40
	G60, L61, A56, T107, K6, T123
	A39, L17, G20, T123, P26, S24
	G60, L61, A56, T107, K55, S57
	G60, L61, A56, T123, K126, S101
	Epi#41
	P19, L17, V3, S1
	P19, L17, V5, S24
	Epi#44
	V65, D64, P66, W92, Y90, A39, V18, P19
	L61, D64, P66, W92, Y90, A39, V18, T91
	Epi#45
	R31, P34, F35, N93, V94
	K96, P34, F35, N93, G32
	Epi#47
	I127, S101, R31, I97, A98, L61, N103, P99, P95
	I28, S101, R31, I97, A98, L61, N103, P99, S57
	Epi#48
	H30, K96, P95, P99, G60
	H30, K96, P34, P19, G20
	H30, K96, P34, P19, V18
	H30, K96, P34, P95, V94
	H30, K96, P34 P19, V3
	E38, K89, P70, P66, V65
	H30, K96, P95, P34, G32
	Q36, K89, P70, P66, V65
	Epi#50
	D69, Y90, W92, P66, P70
	D69, Y90, W92, P34, P95
	D69, Y90, W92, T91, P34
	D69, Y90, W92, V94, P95
	D69, Y90, W92, L37, P19
	Epi#51
	K126, H124, E102, R128, I28, R31, H30
	T123, H124, K126, R128, I28, R31, H30
	D4, H124, K126, R128, I28, R31, H30
	PhI p2:
	Epi#02
	T87, K85, Q61, S38, R34, R67
	T87, K85, Q61, P63, R34, V42
	Epi#03
	K10, A90, I88, Y86
	K10, A18, I88, Y86
	Epi#04
	R34, S38, Q61, K85, T87, I88
	R34, S38, Q61, K85, T87, A90
	Epi#05
	G47, A18, S12, T87, G89, T91, T5, V1
	G73, A29, L69, T27, G50, T53, T45, V42
	G11, A18, L20, T91, G89, A90, T87, I88
	Epi#06
	A93, P94, D79, R34, Q61, P59
	A93, P94, D79, R34, Q61, P83
	A93, P94, D80, R34, Q61, P59
	A93, P94, D79, R34, Q61, P63
	Epi#08
	K10, E9, G11, A18, H16, F54
	K46, E48, G47, A18, H16, F54
	K10, E9, S12, A18, H16, F54
	Epi#09
	L69, T27, G73, N76, R67, V77, D79, R34, A43, T45, V42
	L69, T27, A29, E30, R67, V77, D80, R34, A43, T45, V42
	Epi#10
	D55, A18, N13, S12, F54, G47, K46
	T45, A18, N13, S56, F54, G47, K46
	Epi#09
	L60, S56, E57, D55, K15, N13, S12, G11
	L60, S56, E57, D55, H16, F54, T45, T53
	L60, S56, E57, D55, H16, F54, T45, G47
	Epi#12
	Y86, E84
	Y23, E24
	Epi#18
	N76, R67, F78, V81, A93, Y92, T91, T5, P2, V1
	Epi#19
	D39, W41, S38, Q61, R34, G37
	E40, W41, S38, Q61, R34, A43
	Epi#22
	D79, P94, D80, P83, K85
	D79, P94, D80, P63, K85
	Epi#23
	K10, N13, E14, L60, Q61, P59
	K10, N13, E14, L60, Q61, P83
	K10, N13, E14, L60, Q61, P63
	Epi#24
	E58, K15, E57, P59, S56, E14, Q61
	D55, K15, E57, P59, S56, E58, Q61
	Epi#25
	R34, R67, W41, D39, E40
	Epi#26
	S38, E40, W41, V42, E32, E30
	S38, E40, W41, V42, A43, E32
	Epi#27
	E14, E57, E58, K15
	D55, E14, E84, K85
	Epi#28
	G37, H36, Q61, K85, E84, L60, F54, A43, K46
	G37, H36, Q61, K85, E84, L60, F54, S12, D55
	G37, H36, Q61, K85, E84, L60, F54, S56, D55
	G37, H36, Q61, K85, E84, L60, F54, A43, R67
	G37, H36, Q61, K15, E57, L60, F54, A43, K46
	G37, H36, Q61, K85, E84, L60, F54, S12, K15
	G37, H36, Q61, K85, E84, L60, F54, S56, K15
	G37, H36, Q61, K85, E84, L60, F54, A43, R34
	G37, H36, Q61, K85, E84, L60, F54, A18, D55
	Epi#29
	G73, K72, L69, R67, E30
	I88, N13, L60, F54, E57
	G25, K72, L69, R67, E32
	V77, K75, L69, R67, E30
	G37, H36, L60, F54, E57
	G37, Q61, L60, F54, E57
	Epi#30
	I88, N13, S12, H16, K15, P59, L60
	I88, N13, S56, H16, K15, L60, P59
	I88, N13, A18, H16, K15, P59, L60
	Epi#33
	K46, F54, V42, S56, K15
	H16, F54, V42, S56, K15
	Epi#34
	V1, P2, T5, V4, P94, Y92, T87
	V1, P2, T5, L20, G89, T91, T87
	V81, P94, T5, V1, P2, Y92, T91
	Epi#37
	T27, A29, L69, K72, D26
	A43, R67, L69, K75, N76
	Epi#38
	L20, G89, E9, A18, N13, P59, S56
	Epi#40
	G49, L20, G89, Y86, K85, T87
	G49, L20, G89, T87, K10, S12
	G49, L20, G89, T87, K10, T7
	Epi#44
	V77, R67, D79, P94, Y92, A93, V1, P2
	L69, R67, D79, P94, Y92, A93, V1, T5
	Epi#45
	D79, P94, F78, N76, M74, L69
	D80, P94, F78, R67, D79, V77
	K3, P94, F78, N76, M74, G73
	Epi#46
	A43, R67, R34, P63, H36, Q61
	V77, R67, R34, P63, H36, G37
	L69, R67, R34, P63, G37, Q61
	Epi#47
	G37, E35, E40, A43, R34, L60, N13, P59, S56
	V77, E32, E40, A43, R34, L60, N13, P59, S56
	S38, G37, E40, A43, R34, L60, N13, P59, S56
	Epi#48
	E24, K3, P94, P2, V1
	E84, D80, P94, P2, V1
	Epi#50
	D39, W41, A43, T45
	D39, W41, V42, T45
	Epi#51
	D79, H36, E84, T87, K10, G11, H16
	D39, H36, Q61, K85, P63, R34, W41
	D79, H36, E40, D39, G37, R34, W41
	Q61, H36, E84, T87, K10, G11, H16

TABLE 8
Each row indicates an epitope pattern. At each position (from 1 to
maximum of 12) the cells indicate which amino acids (single letter
coding) are allowed at that position. The last column indicates
the patterns identified using IgE antibody binding.

Epitope

Pattern

Position

Number	1	2	3	4	5	6	7	8	9	10	11	12

1

TS

RQ

YS

NHC

KR

KR

P

HNP

L

IgE

2

RV

R

Y-

PST

FR-

ALPQS-

RKN

ALT

IgE

(SEQ ID

NO: 197)

3

Y

I

AH-

K

L

4

AGIL

ANRTV-

KRY

Q

S

Y-

KR

(SEQ ID

(SEQ ID

NO: 198)

NO: 199)

5

GILVY

STH

ASTR-

G

PT-

RNAFLS

A

G

IgE

(SEQ ID

(SEQ ID

(SEQ ID

NO: 200)

NO: 201)

NO: 202)

6

P

KRQSA

STRC

D

PAN

GA

IgE

(SEQ ID

(SEQ ID

NO: 203)

NO: 204)

7

D

P

AV-

R

S-

D

S-

T

G

8

F

HI-

VA-

FSG-

DE-

KA

IgE

9

NRGLTV-

STAN

ANF

RKH

D-

AILV-

R-

(ENRSV-

AGI-

DGNT-

LIS-

IgE

(SEQ ID

(SEQ ID

(SEQ ID

SEQ ID

(SEQ ID

NO: 205)

NO: 206)

NO: 207)

NO: 208)

NO: 209)

10

KR

RG

F

C-

AST-

RN

NTA

DECT

IgE

(SEQ ID

NO: 210)

11

DE

V-

Q

I

FLK

F

12

E

Y

IgE

13

FWYGL

PG

ALS-

PH

A

T-

P

LRWA

SAH

IgE

(SEQ ID

(SEQ ID

NO: 211)

NO: 212)

14

GV

Q

ILV

I-

Y

GNR

DN

TEH

15

AG

RKQT-

I

D

P

RKN

IgE

(SEQ ID

NO: 213)

16

DN

A

DA

SDN

QRSW

GMR

Y

P

RQL

(SEQ ID

NO: 214)

17

S-

R

S

A

18

VLSFN

AEHNPT-

T-

L-

ST-

Y-

GAL

LIV-

CSF-

R

FRN-

SD

IgE

(SEQ ID

(SEQ ID

NO: 215)

NO: 216)

19

AGLKM

R

Q

QSC

NTW

DEI

IgE

(SEQ ID

NO: 217)

20

D

G

D

KN

L

LF-

P

K

V

A

IgE

21

P

S

I-

I

LR-

CI

IgE

22

EDKW

ACLPTV

D-

ASLPM-

D-

IgE

(SEQ ID

WY-

(SEQ ID

NO: 218)

(SEQ ID

NO: 220)

NO: 219)

23

AP

LQF

SYLN-

E

N

RK

IgE

(SEQ ID

NO: 221)

24

KQ

AELFPR-

TSFR

P

EA

GK

DE

IgE

(SEQ ID

(SEQ ID

NO: 222)

NO: 223)

25

ENV

DE

IW-

RKH

R

IgE

26

DE

AGE

PHV

W

E-

S

W

IgE

27

K

DE

E

DE

IgE

28

DKR

APSG-

QF-

CFIKLW-

E

FIKLW-

Q

DH-

AGILV

IgE

(SEQ ID

(SEQ ID

(SEQ ID

(SEQ ID

NO: 224)

NO: 225)

NO: 226)

NO: 227)

29

E

RF-

L

KRQHNGP

GILV

IgE

(SEQ ID

(SEQ ID

NO: 228)

NO: 229)

30

LVP

LIP

IKLPQS-

H

AS-

LIMN

GI

(SEQ ID

(SEQ ID

NO: 230)

NO: 231)

31

D

FI-

MV-

FW

R

N

QR

L

32

V

f-

DE

A

A

F

33

KR

SA-

S

VP

YF

KQH

IgE

34

STP

STY

GPR-

GLV

STM

WP

IVW

35

I

M

S

A-

L

AG

36

AW

A

PV-

K-

Q-

ST

Y-

G-

V-

A

A

TP

IgE

37

NYD

KR

L

ARV

TYAP

IgE

(SEQ ID

NO: 232)

38

S

P

N

LR-

RV-

AR-

E

G

L

39

L

G

P

RT-

HL-

E

A

40

ST-

APK-

YT

AG

L-

AGR

IgE

41

St

V-

L

Yh-

P-

42

RQ

R

P-

H-

NQG

S

P

L

43

T-

RI

ML

S

HQ

GL

YA

WC

I

44

PT

AGV

SA

Y

W-

P-

D-

RQ-

ILVS

IgE

(SEQ ID

NO: 233)

45

LVG

MD-

RN

Y-

F

PH

KRD

IgE

46

AGQ

HNQGC

P

R

R

AVLCY

IgE

(SEQ ID

(SEQ ID

NO: 234)

NO: 235)

47

PS

RP

N

LFQA-

AR

AILMNV

RE

AGSYLE

LIAGVS

(SEQ ID

(SEQ ID

(SEQ ID

(SEQ ID

NO: 236)

NO: 237)

NO: 238)

NO: 239)

48

GV

P

P

KHQD

SHQE

(SEQ ID

(SEQ ID

NO: 240)

NO: 241)

49

KN

Q-

TMC

WYC-

Q

Q

FP-

VP-

L

W-

D

50

PST

STAPLWV

W

WY-

RHD

(SEQ ID

NO: 242)

51

WH

TSKHRQG

LIRKGP

DSRTQG

DEKQHT

H

RKQDT

(SEQ ID

(SEQ ID

KH- (SEQ

(SEQ ID

(SEQ ID

NO: 243)

NO: 244)

ID NO:

NO: 246)

NO: 247)

245)

52

Q

DNT-

W

R

STRE-

A

FW

(SEQ ID

NO: 248)

Example 10

For this example the third-generation epitope sequences were determined in further 11 environmental allergens (Bosd2, Equc1, Gald4-mutant (with alanine substituted for glycine in position 102), Hevb8, Profillin1-AC, Profillin1-AT, Profillin2-AC, Profillin-birch pollen, Rag weed pollen5 and Vesv5), based on their structures sequences (SEQ ID NOS: 12, 13, 15, 16, 17, 18, 19, 20, 21 and 22, respectively), their structures (1bj7.pdb, 1ew3.pdb, 1flu.pdb, 1g5u.pdb, 1prq.pdb, 1a0k.pdb, 1f2k.pdb, 1cqa.pdb, 1bbg.pdb, and 1qnx.pdb, respectively), and computer modelling of the epitope patterns that had been assembled in our database (shown in Table 8). Further, the epitope sequences of the four environmental allergens of example 9, Bet v1, Der f2, Der p2, and Phl p2, were redetermined.

The additional allergens arise from common sources of allergy: cows (Bos d2 which is a bovine member of the lipocalin family of allergens), horses (Equ C 1, a major horse allergen aslo of the lipocalin family), Hen egg white (Lysozyme Gal D 4), Latex (Hev b8, a profilin from Hevea Brasiliensis), Acanthamoeba castellani (Profilin1-AC, a profilin isoform IA and Profilin2-AC, a profilin isoform II), Arabidosis thaliana (Profillin1-AT a cytoskeleton profilin), Birch (Profilin-birch pollen (Birch pollen profilin), Rag weed pollen5 (Ragweed pollen allergen V from Ambrosia trifida) and whasp venom (Ves v5 allergen from Vespula vulgaris venom).

The protein surface is scanned for epitope patterns matching the given “consensus” sequence of about 6-12 residues. First, residues on the protein surface that match the first residue of the consensus sequence are identified. Within a specified distance from each of these, residues on the protein surface that match the next residue of the consensus sequence are identified. This procedure is repeated for the remaining residues of the consensus sequence. The method is further described under the paragraph “Methods” above and the program can be found in Appendixes.

The critical parameters used in this screening included:

• • i) a maximal distance between the alpha-carbon atoms of subsequent amino acids,
  • ii) a minimal accessability of the amino acid of 20 Å2,
  • iii) the largest maximal distance between the most distinct amino acids should be less than 25 Å,
  • iv) the best epitope were taken,
  • v) the homology with the epitope pattern of interest was 100%

In this way a number of potential epitopes are identified. The epitopes are sorted according to total surface accessible area, and certain entries removed:

• • a. Epitopes that contain the same protein surface residue more than once. These are artefacts generated by the described algorithm.
  • b. Epitopes which are “too big”, i.e. where a distance between any two residues in the epitope exceeds a given threshold.

The epitope sequences found were:

Bosd2:
Epi#01
L65, P155, P156, R17, R40, N37, Y39, R41, T67
L65, P155, P156, R17, R40, N37, Y39, R41, S52
L64, P155, P156, R17, R40, N37, Y39, R41, T54
Epi#02
T121, K150, S122, R17, P156, Y39, R41, R40
T121, K150, S122, R17, P156, Y56, R36, V30
Epi#03
L128, K130, H92, I7, Y76
L134, K130, H92, I7, Y76
L128, K130, H92, I91, Y76
Epi#04
R72, Y76, S50, Q73, K71, V69, I45
K71, Y76, S50, Q73, R72, V69, L80
K71, Y76, S50, Q73, R72, V69, I42
Epi#06
G14, P13, D47, S10, K11, P9
G14, P13, D47, S10, S94, P9
G14, P13, D47, C44, S10, P9
Epi#08
K71, E49, S50, V69, F82
K71, E49, S50, V79, F82
Epi#09
I7, S10, D8, E95, K119, N96, S122, T121
S10,I7, D8, E95, K11, N96, S122, T124
Epi#10
E15, T54, R41, T67, F55, R17, K119
E43, T54, R41, T67, F55, R17, K119
E31, T151, N153, C63, F55, R40, R41
E31, TI51, N153, C154, F55, R41, R17
Epi#11
K26, I145, Q132, E143
K26, I145, Q132, E137
K26, I145, Q132, E129
Epi#12
Y105, E108
Y83, E81
Epi#15
N153, P156, D152, I149, T121, G120
R17, P156, D152, I149, T121, G120
N153, P156, D152, I149, R17, G14
Epi#18
R109, I110, G107, Y83, T85, E81, V69
R109, I110, G107, Y105, T85, E81, V69
Epi#19
E43, N46, S50, Q73, R72, K71
D47, N46, S50, Q73, R72, G75
E49, N46, S50, Q73, R72, K71
I45, N46, S50, Q73, R72, K71
Epi#20
V30, K28, P34, L57, L65, K58, D59, G32, D27
V30, K28, P34, L57, L64, K58, D59, G33, D27
Epi#22
D8, S10, D47, P13, E15
D8, S10, D47, P13, E43
D47, S10, D8, V93, E95
D8, S10, D47, C48, K71
Epi#23
k119, N96, E127, S122, L128, P125
K150, N147, E146, Y20, F123, P125
K11, N96, E127, S122, L128, P125
Epi#24
E129, K130, E126, P125, S122, L128, Q133
E126, K130, E129, P125, S122, R17, K119
E126, K130, E129, P125, T124, L128, Q133
Epi#25
R72, K71, I45, D47, N46
R72, K71, I45, E43, N46
Epi#27
D47, E49, E74, K71
D24, E143, E146, K150
D47, E43, E15, K119
Epi#28
L134, Q133, L128, E126, K130, F123, S122, K150
Q132, K130, E126, L128, F123, S122, K150
L65, D59, Q60, K58, E31, L57, G32, D27
G61, D59, Q60, K58, E31, K28, G32, D27
Epi#29
V69, K71, L80, R72, E74
I45, K71, L80, R72, E74
G61, Q60, L64, F55, E68
Epi#30
G120, N96, S94, H92, K130, L128, P125
I91, I7, S94, H92, K130, L128, P125
Epi#33
K130, F123, P125, S122, K150
k71, Y76, P9, S10, S94, K119
Epi#34
I7, P9, S10, G14, R17, T121, S122
I45, P13, S10, G14, R41, Y39, P156
Epi#37
T67, V69, L80, K71, Y76
P156, R40, L65, K58, D59
P155, R40, L65, K58, N153
Epi#38
L80, G84, E108, R109, N25, P141, S136
Epi#39
E137, R138, P141, G139, L134
E31, L57, R36, P34, G84, L80
Epi#40
R17, G120, T121, K150, S122
R17, G120, T121, K150, T151
Epi#41
P34, Y83, L80, V69, S52
P34, Y83, L80, V79, S50
Epi#42
L128, P125, S122, G120, R17, R41
L128, P125, S122, G120, R17, R40
Epi#44
S10, D47, P9, Y76, S50, V69, T67
I45, D47, P9, Y76, S50, V69, T67
Epi#45
D27, P34, F82, Y105, R109, D106, G107
D59, P34, F82, Y105, R109, D106, G107
K58, P34, F82, Y105, R109, D106, G107
D27, P34, F82, Y105, R109, D106, G84
Epi#46
Y39, R41, R40, P155, C63, Q60
Y20, R17, R40, P155, C63, Q60
Epi#47
L128, E126, E129, L134, R138, Q133, N142, P141, S136
V69, E81, E68, I42, R41, F55, N37, R40, P156
V69, E43, E15, I42, R41, F55, N37, R40, P156
S122, E127, E129, L134, R138, Q133, N142, P141, S136
Epi#48
E43, D47, P13, P9, V93
S10, D47, P9, P13, G14
E43, D47, P13, P9, V90
E49, D47, P13, P9, V93
Equc1:
Epi#02
L66, N68, A65, F90, S69, Y72, R64, V89
A65, R64, S31, F28, S112, Y123, R110, V108
L179, R180, Q178, F177, P143, Y38, R141, V145
L66, R64, S31, F28, S112, Y123, R110, V125
L66, N68, A65, F90, S69, Y72, R64, V62
Epi#03
K32, A65, I63, Y72
Epi#05
G35, A65, S69, T93, G97, R26, S112, Y123
G35, A65, S69, T93, G97, R26, S112, I25
Epi#07
G97, T93, S70, D91, S100, R110, V125, P132, D128
Epi#08
K129, D130, F127, V108, F90
K129, D130, F127, V108, F109
K129, D130, F127, V125, F136
K129, D130, F127, V125, F133
Epi#10
E48, N53, N80, T77, C83, F177, R175, K172
E82, N80, N53, T77, C83, F177, G181, R180
E52, N53, N80, T77, C83, F177, R175, K172
Epi#11
F133, K47, I167, Q158, V163, E165
Epi#12
Y38, E142
Y38, E36
Y139, E142
Epi#13
K129, P132, D45, I167, Q158, G161
R131, P132, D45, I167, K164, G161
Epi#16
P87, Y72, R64, S70, S69, D67, A65, N68
Epi#17
A65, S31, R64, S34
Epi#18
R64, S31, I30, A65, S34, L66, N68, S69
Epi#19
E82, N80, C83, Q178, R175, K172
Epi#22
D130, P132, D128, Y106, K129
Epi#23
D144, K150, E148, P147, S146, E151, K155
Epi#25
R160, K159, I156, E151, E148
Epi#27
E118, E142, D144, K172
E36, E142, D144, k172
Epi#28
I173, D174, Q178, L179, E85, C83, F177, G181, R180
I173, D174, Q178, L179, E85, C83, F177, P143, D144
Epi#29
G181, Q178, L179, R180, E36
G181, Q178, L179, R180, E85
Epi#30
I30, N27, S112, H119, I121, I25, V23
Epi#31
L122, R110, N27, R26, F28, I30, D96
L124, R110, N27, R26, F28, I30, D96
Epi#33
H119, Y38, V62, S34, S31, R64
Epi#34
V62, P87, M88, V89, R64, S31, S34
Epi#37
P87, V89, L66, R64, D67
Epi#40
R64, L66, A65, Y72, S34
R64, L66, A65, Y72, S69
Epi#41
P132, Y106, L101, V89, S100
P132, Y106, L101, V89, S70
Epi#44
V46, R131, D128, P132, Y106, S100, V89, P87
Epi#45
K129, P132, F127, Y106, N102, D91, V89
K129, P132, F127, Y106, N102, D104, G105
Epi#47
S146, E148, E152, V23, R26, A24, N27, R110, S112
V23, E115, E118, N116, R26, F28, N27, R110, S112
Gald4:
Epi#01
L75, N65, P70, R73, R61, N59, Y53, R45, T47
L75, N65, P70, R68, R61, N59, Y53, R45, T47
Epi#02
A90, N77, L75, R73, P70, R61, R68
A122, R125, Q121, T118, R114, R112
Epi#04
R21, Y20, S24, Q121, R125, R128, L129
R21, Y20, S24, Q121, R125, R128, G126
Epi#05
G16, A10, R128, G126, A122, T118, G117
G4, A10, R128, G126, A122, T118, G117
Epi#06
G67, P79, D66, R61, R73, P70
G67, N65, D66, S72, R73, P70
G49, N46, D48, R61, R73, P70
Epi#07
G71, T69, D66, S72, R73, P70, D48
G67, T69, D66, S72, R73, P70, D48
Epi#08
k1, D87, S86, V2, F38
K1, D87, S86, V2, F3
Epi#09
Epi#10
E7, A11, R14, A10, C6, F3, R5, R125
D87, A11, R14, A10, C6, F3, R5, R125
T47, N46, N44, S36, F34, R114, R112
D18, A10, R14, A11, C6, F3, R5, R125
T118, N113, R112, A100, F34, R114, K116
Epi#11
L129, I124, Q121, V120, D119
Epi#12
Y53, E35
Epi#15
R73, P70, D66, I78, A82
R73, P70, D66, I78, A90
Epi#17
A102, S100, R21, S24
Epi#18
R112, N113, R114, F34, V109, A107, A102, N103
N113, R112, R114, F34, V109, A107, N103, S100
Epi#19
D18, N19, S24, Q121, R125, L129
D18, N19, S24, Q121, R125, G126
Epi#22
D48, P70, D66, W63, W62
D66, P70, D48, T69, W62
D48, P70, D66, W63, K97
Epi#23
R45, N44, E35, N39, Q41, A42
R45, N44, E35, Y53, Q41, A42
Epi#25
R128, R125, W123, D119, N27
R128, R125, W123, D119, V120
Epi#26
W62, S72, W63, P79, A82, D87
W62, S72, W63, P79, G67, D66
Epi#28
G117, D119, Q121, I124, E7, C6, F3, A11, R14
A122, D119, Q121, I124, E7, C6, F3, A11, R14
Epi#29
G126, R125, L129, R128, E7
G16, R14, L129, R128, E7
Epi#30
I124, L129, A10, H15, I88, L84
I124, L129, A11, H15, I88, L84
Epi#31
L75, R73, N65, R61, W62, I98, D101
L75, R73, N74, R61, W62, I98, D101
Epi#33
Q41, F38, V2, S86, S85, K1
Q41, F38, V2, S36, A110, R114
Epi#34
W63, W62, T69, G71, R73, S72, P70
W62, W63, S72, L75, R73, T69, P70
Epi#36
A110, A107, A102, S100, K96, A90, A82
Epi#37
A10, R128, L129, R14, D18
A10, R128, L129, K13, N19
Epi#40
R128, L129, A11, T89, A90, S85
R14, L129, A11, T89, A90, S85
Epi#41
Y53, L84, S81
Y53, L84, S86
Epi#42
P79, S81, N65, P70, R61, R73
P79, S81, N65, P70, R61, R68
Epi#44
L129, R14, D18, Y20, S24, V120, T118
L129, R14, D18, Y23, S24, V120, T118
Epi#46
L75, R61, R73, P70, N65, G67
L75, R73, R61, P70, N65, A82
L75, R61, R68, P70, N65, G67
Epi#47
S72, G71, R68, N65, R61, L75, N77, R73, P70
G67, S72, R68, N65, R61, L75, N77, R73, P70
Epi#49
D87, L84, Q41, Q57, Y53, T43, N44
D87, L84, Q57, Q41, Y53, T43, N46
D87, L84, Q41, Q57, Y53, T43, N39
Epi#50
R73, W62, W63, P79, S81
R73, W63, W62, S72, P70
Epi#51
D18, H15, K13, R14, L129, R125, W123
Epi#52
F34, A110, R112, R114, W111, N27, Q121
F3, A11, E7, R5, W123, D119, Q121
W123, A122, T118, R114, W111, N27, Q121
Hevb8:
Epi#01
L20, P109, P112, k86, R84, N116, Y125, Q129, T111
L110, P109, P112, K86, R84, N116, Y125, Q129, T111
Epi#02
A48, K43, Q41, F42, T70, Y72, R84, V74
T21, R19, P109, P112, R84, V74
A49, K43, Q41, F42, T70, Y72, R84, V74
Epi#03
L65, K86, I75, Y72
Epi#05
G30, A48, L60, P62, G58, T63, H66, G69
G58, A61, R84, P112, G113, T111, S89, G88
G80, A81, F54, P79, G58, T63, H66, G69
G77, A81, F54, P79, G58, T63, H66, G69
Epi#06
G58, P79, D55, S59, K52, P57
G80, P79, D55, S59, K52, P57
G77, P79, D55, S59, K52, P57
Epi#07
G17, T5, S2, D16, R19, P109, D107
Epi#08
K52, D45, S44, A49, H66, F42
Epi#10
E78, A81, R96, F54, G58, K52
D55, A81, R96, F54, G80, K52
Epi#11
F54, L60, I83, Q76, V82, E78
Epi#12
Y106, E108
Epi#13
H66, L65, P62, T63, A61, P57, A81, P79, G58
H66, L65, P62, T63, A61, P57, A81, P79, G80
H66, L60, P62, T63, A61, P57, A81, P79, G77
Epi#15
R19, P109, D107, I105, K86, G88
Epi#18
R19, G17, P109, S89
Epi#22
D29, S44, D45, A48, K52
D29, M51, D55, P79, E56
D45, M51, D55, P79, E78
D29, S44, D45, A49, K52
D45, M51, D55, P79, E56
D29, M51, D55, P57, E78
D29, M51, D55, P57, E56
D45, M51, D55, P57, K52
D45, M51, D55, P57, E78
Epi#24
D55, K52, E56, P79, F54, E78, Q76
D45, K52, E56, P57, F54, E78, Q76
Epi#25
R84, K86, I105, D107, E108
R96, H28, I26, D29, V3
Epi#26
W33, S2, W3, V32, G30, D29
Epi#27
D53, E56, D55, K52
Epi#28
V32, Q41, K43, E46, K52, F54, P57, D55
G69, Q41, K43, E46, K52, F54, P57, D55
Epi#29
G130, Q99, L127, R96, E78
L127, Q99, L131, R96, E78
G98, Q99, L127, R96, E78
Epi#30
G69, L67, A49, H66, K71, L65, P62
G80, M51, A48, H28, Q99, L127, L131
Epi#33
Q41, F42, V32, S31, S44, K43
Q41, F42, V47, S44, A48, K52
Q41, F42, V47, S44, A49, K52
Epi#34
I105, P112, S89, L110, R19, T21, S37
I105, P112, T111, L20, R19, T21, S37
Epi#37
T63, A49, L60, K52, D55
P62, V74, L60, K52, D45
P62, A61, L60, K52, D55
Epi#38
G77, E78, R96, V82, R84, N116, P112, S89
Epi#39
A48, E46, H66, T63, P62, G58, L60
A49, E46, H66, T63, P62, G58, L60
Epi#40
R19, L110, G113, T111, P109, S89
R19, L110, G113, T111, P112, S89
Epi#41
P62, L65, V47, S44
P109, Y106, L110, S89
P112, Y106, L110, S8
Epi#44
L20, R19, D16, W3, Y6, S2, G17, P109
L110, R19, D16, W3, Y6, S2, G17, P109
Epi#45
K52, P57, F54, R96, D124, L127
D55, P79, F54, R96, D124, L131
Epi#47
I75, G77, E78, V82, R84, N116, P112, S89
I75, G77, E78, I83, R84, N116, P112, P109
Epi#48
E78, Q76, P79, P57, G58
E78, Q76, P79, P57, G80
E78, Q76, P79, P57, G77
Epi#50
D9, W3, W33, S2, T5
D16, W3, W33, S2, T5
Epi#51
R19, H18, E108, S89, K87, K71, H66
R19, H18, E108, D107, K87, K71, H66
Profillin1-AC:
Epi#01
L116, N111, P106, K80, K81, N101, S83, Q105, T108
L116, N111, P106, K80, K81, N101, Y100, Q105, S83
Epi#02
T44, N51, P54, R56, T69, Y78, R71, V68
L24, K93, S92, R75, S76, Y78, R71, R56
Epi#03
L24, K93, I121, Y119
L24, K90, I121, Y119
Epi#04
K80, Y100, S83, Q105, K103, N101, G82
K80, Y100, S83, Q105, K103, T17, G12
K80, Y100, S83, Q105, K103, T17, G14
Epi#05
G34, A33, A36, T38, G64, A63, H66, V68
G34, A33, S32, T17, G12, T4, S1, Y5
Epi#06
A46, N0, D53, R56, A57, P54
A52, N50, D53, R56, A57, P54
A72, N50, D53, R56, A57, P54
A57, P54, D53, S47, Q43, P39
Epi#07
G64, T38, D61, S58, R56, A57, P54, D53
G64, T38, D61, S58, R56, A52, P54, D53
Epi#08
K103, E102, G82, V68, H66, F60
K81, E102, G82, V68, H66, F60
Epi#09
L24, S47, D53, A57, V68, R71, L70, R56, N51, N50, R75
L24, S47, D53, A57, V68, R71, L70, R56, N51, T44, T38
Epi#10
D74, N50, N51, T44, F60, R56, R71
D53, N50, N51, T44, F60, R56, R71
Epi#11
F125, K93, I121, Q123, D118
F125, K90, I121, Q123, D118
F49, K90, I121, Q123, D118
Epi#12
Y119, E114
Y100, E102
Epi#13
A57, R56, P54, T44, A40, P39, A36, G64, Y67
S58, A57, P54, T44, A40, P39, A36, G64, Y67
Epi#15
N51, P54, D53, I55, R56, A57
R56, P54, D53, I55, T69, A57
R56, P54, D53, I55, T44, A40
Epi#16
Q105, P106, Y100, G14, Q18, S32, A36, A33, D7
Q105, P106, Y100, G14, Q18, S32, A36, A63, D61
Epi#17
A110, S76, R75, S92
A72, S76, R75, S92
Epi#18
N51, N50, R75, S92, L24, S47, T44, P39, N27
N51, N50, R75, S92, L24, T28, T38, P39, N27
Epi#22
D53, S47, D25, L24, K93
D53, S58, D61, V68, K81
Epi#23
K103, N101, E102, S83, Q105, P106
K103, N101, E102, S83, Q105, A84
Epi#24
E114, K115, A110, P106, S83, E102, K103
D53, G59, A57, P54, R56, L70, K80
E102, K103, A15, P106, S83, A84, Q105
Epi#25
R71, R56, I55, D53, N50
R71, R56, I55, D53, N51
Epi#28
I104, Q105, K103, E102, K81, S83, K80
G107, Q105, K103, E102, K81, G82, K80
A84, Q105, K103, E102, K81, S83, K80
A110, Q105, K103, E102, K81, S83, K80
Epi#29
I121, K115, L116, E114
V112, K115, L116, E114
Epi#30
G59, I55, S58, H66, K80, L70, V68
G59, I55, S58, H66, K80, P106, V99
Epi#33
K80, Y78, V68, S58, A57, R56
K81, Y67, V68, S58, A57, R56
Epi#34
I55, P54, S58, V68, R71, Y78, P106
W29, W2, T4, V11, G12, Y5, S1
Epi#36
A63, A36, A33, V11, G14, Y100, S83, Q105, K103, P106, A110, A15
A63, A36, A33, V11, G14, Y100, T108, Q105, K103, P106, A15, A110
Epi#37
A57, R56, L70, R71, Y78
A57, V68, L70, R56, D53
Y78, R71, L70, R56, N51
P54, R56, L70, R71, D73
T69, R71, L70, R56, D53
Epi#38
G82, E102, A84, V99, N101, P106, S83
Epi#40
R71, L70, A72, Y78, K80, S83
R71, L70, G59, T69, K81, S83
R56, L70, A72, T69, K81, S83
Epi#41
P106, Y78, L70, V68, S58
Epi#42
P54, S47, N51, R56, R71
P54, S58, G59, R56, R71
Epi#44
S83, Q105, P106, Y78, A110, G107, T108
V68, R71, D73, Y78, A110, G107, T108
L70, R71, D73, Y78, A110, V112, T108
L70, R71, D73, Y78, A110, G107, P106
Epi#45
K81, H66, F60, R56, D53, G59
K80, H66, F60, R56, D53, G59
D61, H66, F60, R56, D53, G59
Epi#46
L70, R71, R56, P54, N51, A52
L70, R71, R56, P54, N51, A72
V68, R71, R56, P54, N51, A46
Y78, R71, R56, P54, G59, A57
Epi#47
V68, A57, R56, L70, R71, A52, N51, P54, S58
S58, A57, R56, L70, R71, A72, N51, P54, S47
Epi#49
D25, L24, Q43, Q41, T44, N51
D25, L24, Q43, Q41, T38, N27
Epi#50
D7, W2, W29, S1, T4
D7, Y5, W2, W29, S1
Epi#51
K80, H66, D61, T44, P39, T28, W29
K80, H66, D61, T38, P39, T28, W29
Profillin1-AT:
Epi#01
P109, P89, K86, R84, N116, Y106, Q114, T111
Epi#02
L42, K43, Q45, F66, T63, Y72, R84, V74
L42, K43, Q45, F66, T63, Y72, R84, V82
Epi#03
K96, I127, Y125
K86, I75, Y72
Epi#05
G77, A81, F54, P57, G58, A61, T63, V74
G58, A61, F59, P57, G77, A81, T97, G80
G80, A81, F54, P57, G58, A61, T63, Y72
Epi#06
G17, P109, D107, T21, K38, P40
G112, P109, D107, T21, K38, P40
G88, P89, D107, T21, K38, P40
Epi#08
K52, E55, G58, V74, F66
K51, E55, G58, A61, F59
Epi#09
D29, D48, K52, F59, A61, T63
D29, D48, K51, F59, A61, T63
Epi#10
E108, T111, N18, T21, F39, G68, K71
E108, T111, N18, T21, F105, G112, K86
Epi#11
F105, K86, I75, Q76, V82, E78
F66, K43, I47, Q28, V32, D29
F59, K52, I47, Q28, V32, D29
Epi#12
Y125, E130
Y125, E128
Epi#15
K43 P44, D29, I47, K52, G58
K43, P44, D48, I47, Q45, G49
K43, P44, D29, I47, K51, G80
Epi#20
K38, P40, F39, L42, K43, D48, G30, D29
K51, P57, F59, L60, K52, D48, G30, D29
Epi#22
D48, P44, D29, V32, W33
D48, P44, D29, V32, W3
Epi#24
D29, K51, E56, P57, F59, E55, Q79
D48, K52, E55, P57, F59, E56, Q79
Epi#25
R121, K95, I83, D53, E55
R121, K95, I83, E78, V82
Epi#26
W33, S2, W3, V32, G30, D29
Epi#27
E128, E130, D124, K96
E130, E128, D124, K95
Epi#28
I75, Q76, E78, Q79, P57, K51
A61, Q76, E78, Q79, P57, K52
V32, D29, Q99, E130, I127, S129, D124
V32, D29, Q99, I127, E128, S129, D124
Epi#29
V32, Q41, L42, F66, E70
G69, Q41, L42, F66, E70
G68, Q41, L42, F66, E70
Epi#30
G17, N18, H19, Q114, L117, V15
G17, M110, H19, Q114, L117, V15
G113, M110, H19, Q114, L117, V15
Epi#33
Q41, F39, P40, S36, A37, K38
Epi#34
V74, P62, M73, G88, P89, Y106, T111
Epi#37
T111, V15, L117, R121, Y125
T111, V15, L117, R121, D124
Epi#39
A81, E55, P57, G58, L60
A81, E78, P57, G58, L60
Epi#40
R121, L117, G112, Y106, P109, T111
R121, L117, G112, Y106, P89, T111
Epi#41
Y125, L131, S129
Epi#44
I75, R84, Y72, A61, G58, P62
I75, R84, Y72, A61, V74, T63
Epi#45
K38, P40, F105, Y106, N18, D14, G17
K38, P40, F105, Y106, N18, D107, G88
K38, P40, F105, Y106, N18, D14, V15
Epi#48
E16, H19, P109, P89, G88
E16, H19, P109, P89, G112
Epi#49
D124, L131, Q99, Q28, T97, N98
D124, L131, Q99, Q28, T97, K96
Epi#50
D9, Y6, W3, W33, S2
D9, W3, W33, S2, S5
D9, W3, W33, V32, S31
Epi#51
D14, H19, E108, T111, L117, R121, H10
D107, H19, E16, Q114, L117, R121, H10
D14, H19, D107, T21, K38, Q35, W33
Profillin2-AC:
Epi#01
L116, N111, P106, K80, K81, N101, S83, Q105, T108
L116, N111, P106, K80, K81, N101, S83, Q105, T108
Epi#02
T53, N58, S57, R56, T69, Y67, R66, V68
T53, K50, A52, R56, T69, Y67, R66, V68
T53, K50, A72, R56, T69, Y67, R66, V68
Epi#03
L116, K115, I121, Y119
Epi#04
K81, Y100, S83, Q105, K103, T17, G12
K80, Y100, S83, Q105, K103, A84, G82
K81, Y100, S83, Q105, K103, T17, G14
K80, Y100, S83, Q105, K103, N101, I104
K81, Y100, S83, Q105, K103, A15, G107
Epi#06
A54, N47, D25, T28, A36, P39
A40, N27, D25, T28, A36, P39
A44, N47, D25, T28, A36, P39
G34, A33, D7, T31, A36, P39
A43, N47, D25, T28, A36, P39
Epi#08
K103, E102, G82, V68, F60
K103, E102, G82, V68, F60
K81, E102, G82, V68, F60
Epi#10
T53, N58, R56, S57, F60, R66, K81
E61, N58, R56, S57, F60, R66, K80
Epi#11
F125, K93, I121, Q105, E102
F125, K93, I121, Q123, D118
Epi#12
Y100, E102
Y119, E114
Epi#13
A52, A44, P39, A43, H24, S92, G124, Y119
A46, A44, P39, A43, H24, S92, G124, Y119
Epi#15
K103, P106, D118, I121, K93, G124
K103, P106, D118, I121, Q105, G107
K103, P106, D118, I121, Q123, G122
Epi#16
Q105, P106, Y78, R71, S57, N58, A54, A44, D51
Q105, P106, Y78, R71, R56, D51, D74, A52, N47
Epi#18
R66, N58, R56, S57, V68, G82, S83, E102, N101
R66, N58, R56, S57, V68, G82, S83, P106, N101
Epi#22
D74, A52, D51, T53, K50
D25, A44, D51, T53, K50
D74, A46, D51, T53, K50
D74, A72, D51, T53, K50
Epi#23
K103, N101, E102, S83, Q105, P106
K103, N101, E102, S83, Q105, A84
Epi#24
D74, K81, A84, P106, S83, E102, K103
D74, K81, E102, P106, T108, A15, K103
Epi#25
R66, K81, E102, N101
Epi#28
I121, D118, Q105, K103, E102, K81, G82, D74
G107, D118, Q105, K103, E102, K81, G82, D74
G122, D118, Q105, K103, E102, K81, G82, D74
Epi#29
I121, K115, L116, E114
V112, K115, L116, E114
Epi#30
I55, N47, A44, H24, K93, I121, L116
I55, N47, A43, H24, K93, I121, L116
Epi#31
R56, N58, R66, F60, V68, I55, D51
R66, N58, R56, F60, V68, I55, D51
Epi#33
K115, Y119, P106, S83, A84, K103
Q123, Y119, P106, S83, A84, K103
K81, Y67, V68, S57, A54, R56
K80, Y78, V68, S57, A54, R56
Epi#34
W29, W2, T8, V11, G12, 4, S1
W29, W2, T4, G12, G14, T13, T8
Epi#37
T108, V112, L116, K115, Y119
T108, A110, L116, K115, N111
T13, V112, L116, K115, D118
P106, A110, L116, K115, N111
Epi#38
G64, E61, A40, V37, N27, P39, S38
G82, E102, A84, V99, N101, P106, S83
Epi#39
A110, E114, T108, P106, G122, L116
Epi#40
G14, G12, T17, K103, S83
R56, A52, T53, A54, S57
R66, A63, T65, K81, S83
R56, A72, T53, A54, S57
R56, G59, T53, A54, S57
R66, G64, Y67, K81, S83
Epi#42
P106, S83, G82, R75, R71
Epi#44
S1, Q3, D7, W2, Y5, S32, G12, T8
S1, Q3, D7, W2, Y5, A30, A36, P39
S1, Q3, D7, W2, Y5, S32, V11, T8
S1, Q3, D7, W2, Y5, S32, G12, T4
S1, Q3, D7, W2, Y5, A30, A33, T31
S1, Q3, D7, W2, Y5, A30, A36, T28
S1, Q3, D7, W2, Y5, S32, G12, T13
S1, Q3, D7, W2, Y5, S32, G34, T31
Epi#45
K93, H24, F49, R75, D74, G82
D25, H24, F49, R75, D74, G82
Epi#47
A36, G64, E61, A40, A44, A54, N58, R56, S57
Epi#50
D7, Y5, W2, T8, S1
D7, W2, W29, T28, P39
Epi#51
K90, H24, K93, D25, P39, T28, W29
T91, H24, K93, D25, P39, T28, W29
Profillin-brich pollen:
Epi#01
L124, N118, P114, K88, K73, H68, Y74, R86, T95
Epi#02
T113, N118, Q116, P114, R86, V76
T50, K54, L62, T65, Y74, R86, V84
Epi#03
L133, K98, I129, Y127
Epi#04
S40, Q43, K45, T50, G32
S40, Q43, K45, T50, G51
S40, Q43, K45, T50, I49
Epi#05
G82, A81, A83, P59, G60, A63, T65, V76
G82, A83, A81, P59, G60, A63, H61, V76
G79, A81, A83, P59, G60, A63, T65, V76
Epi#06
G70, P46, D31, T50, K54, P59
A81, P59, D55, T50, Q47, P46
G32, P46, D31, T50, K45, P42
G51, P46, D31, T50, K54, P59
Epi#08
A81, E57, G60, A63, H61, F56
A81, E57, G60, V76, H68, F44
K54, E57, G60, A63, H61, F56
Epi#11
F56, K98, I85, Q78, V84, E122
F56, K98, I27, Q37, V34, D31
F56, K97, I85, Q78, V84, E80
Epi#12
Y6, E9
Y127, E122
Epi#13
H68, L62, P64, T65, A63, P59, A81, G82, G79
H61, L62, P64, T65, A63, P59, A81, G79, F56
H68, L62, P64, T65, A63, P59, A83, G79, G60
Epi#15
K45, P46, D31, I49, Q47, G32
K45, P46, D31, I49, K54, G60
K45, P46, D31, I49, K54, G82
K45, P46, D31, I49, T50, G51
Epi#16
Q116, P114, Y108, M12, S39, S40, A23, A24, D8
Q116, P114, Y108, M12, Q37, S40, A23, A24, D8
R86, P114, Y108, M12, S39, S40, A23, A24, D8
Epi#22
D126, L133, D130, Y127, E122
D130, L124, D126, Y127, E122
D130, L128, D126, Y127, E122
Epi#23
R123, N118, E122, L124, L11, A23
R123, N118, E122, L124, L11, A36
R123, N118, E122, L124, L11, A24
Epi#24
E109, G90, E110, P114, R86, E80, Q78
E57, K54, E58, P59, F56, A81, Q78
E58, G60, E57, P59, F56, E80, Q78
Epi#25
R86, K88, I107, E109, E110
R86, K88, I77, E80, V84
R86, K88, I107, E109, V112
Epi#27
57, E58, D55, K54
D55, E57, E58, K54
Epi#28
V34, D31, Q101, K98, E122, L128, Q131, G132, D130
I129, D126, Q131, L128, E122, K98, Q101, G100, D130
I72, H68, Q47, F44, E48, K45, Q43, G70, K73
I72, H68, Q47, I49, E48, K45, Q43, G71, K73
Epi#29
I129, Q101, L128, R123, E122
G132, Q131, L128, R123, E122
Epi#30
I77, M75, A63, H61, P59, L62, P64
G90, M75, A63, H61, K54, L62, P64
Epi#33
Q116, Y108, P111, S91, K89
K88, Y108, P111, S91, K8
Epi#34
V76, P64, M75, L62, G51, T50, P46
I27, W35, S33, V34, G32, T50, P46
V76, P64, T65, L62, G51, T50, P46
Epi#35
A24, L22, A23, S39, M12, I107
A23, L11, A36, S39, M12, I10
Epi#37
Y127, R123, L124, K97, N118
Y108, A23, L11, R123, Y127
A23, A24, L11, R123, Y127
Epi#39
A81, E57, H61, T65, P64, G60, L62
A81, E58, H61, T65, P64, G60, L62
Epi#40
R123, L11, A23, Y108, P111, S91
R123, L11, A24, Y108, P111, T113
Epi#41
P111, Y108, L22, V112, S91
P114, Y108, L22, V112, S91
Epi#43
I27, W35, A36, L11, Q37, S39, M12, I107, T95
Epi#44
I77, R86, P114, Y108, S91, V112, P111
V120, Q116, P114, Y108, S91, V112, P111
L22, Q116, P114, Y108, S91, V112, T113
L22, Q116, P114, Y108, A23, V112, P111
Epi#47
I129 Y127, E122, M119, R123, L124, N118, R86, P114
L133, Y127, E122, M119, R123, L124, N118, R86, P114
Epi#48
E122, Q116, P114, P111, V112
S91, K88, P114, P111, V112
Epi#50
H10, Y6, W3, S2, T5
H10, Y6, W3, T5, S39
Epi#51
K73, H68, K45, Q47, P46, S33, W35
Q101, H30, D31, T50, K45, Q47, H68
Rag weed pollen5:
Epi#03
L4, K37, A33, I34, Y17
L4, K37, A33, I34, Y29
Epi#05
A33, N36, T40, G3, S20, L4
A33, N38, T40, G3, S20, Y25
A33, N36, G3, T40, S20, I22
Epi#06
A33, N36, D2, C19, K24, P21
A33, N38, D2, S20, K24, P21
Epi#09
I22, L4, D2, N38, D1, K37, A33, N36, T40
T9, G15, E7, V14, D30, K32, N36, T40, L4
T9, G15, E7, V14, D30, K32, N38, N36, L4
Epi#12
Y17, E7
Y6, E7
Epi#20
V27, K24, P21, L4, K37, D2, G3, D1
V27, K24, P21, L4, N36, D2, G3, D1
Epi#22
D1, D2, L4, K37
D1, D2, P21, K24
D2, L4, T40, D1
Epi#23
N10, E7, Y6, L4, P21
Epi#25
K32, I34, D30, V14
K37, I34, D30, V14
K16, I34, D30, V14
Epi#33
K32, Y17, V27, S20, K24
K16, Y6, P21, S20, K24
Epi#34
I22, P21, S20, V27, G12, Y17, T9
I22, P21, S20, V27, G12, Y29, S31
Epi#40
G12, G15, Y29, K37, T40
G15, G12, Y17, K16, T9
G12, G15, Y29, K32, S31
Epi#41
P21, Y6, L4, S20
Epi#44
L4, D2, P21, Y25, S20, V27, T40
L4, D2, P21, Y25, S20, G3, T40
Vesv5:
Epi#01
L59, P67, P65, K143, K144, N64, Y140, R62, T61
L59, P67, P70, R57, K204, N73, Y201, Q202, T203
L59, P67, P69, R57, K72, N73, Y201, Q202, T203
L152, N149, P142, K145, K143, N64, Y140, R62, T61
Epi#02
L9, K7, Q108, P191, Y107, R102, V13
L9, K7, Q108, S192, Y107, R102, V13
Epi#03
L9, K7, A105, I6, Y3
Epi#04
K106, Y107, S192, Q108, K7, A105, I6
K106, Y107, S192, Q108, K7, V13, G12
Epi#05
G58, A56, R57, P69, G66, R62, T61, L59
G58, A56, R57, P69, G63, R62, T61, L59
Epi#06
G66, N64, D139, R62, K138, P67
G66, N64, D139, R62, K138, P65
G63, N64, D139, R62, K138, P67
Epi#08
K145, E199, S147, F151
K196, E198, S147, F151
K144, E199, S147, F151
Epi#09
L152, D150, S147, K144, N64, T61, L59
L152, D150, D139, K153, F151, S147, N197
D139, N64, R62, D135, K153, F151, S147, N197
Epi#10
E199, N197, N194, S147, F151, G148, K143
E199, N197, N194, S147, F151, G148, K196
E199, N197, N194, S147, F151, G148, K145
Epi#11
K179, I176, Q177, V30, E178
K29, I176, Q177, V30, E178
Epi#12
Y201, E199
Epi#13
S147, L200, P142, T203, A56, P70, L59, P67, G66
S147, L200, P142, T203, A56, P69, L59, P67, G58
S147, L200, P142, T203, A56, P70, L59, P67, G63
S147, L200, P142, T203, A56, P69, L59, P67, Y140
Epi#15
K106, P191, D103, I6, K5, A105
K106, P191, D103, I6, K7, G12
Epi#16
R57, P70, Y201, M74, Q53, N76, D50, A56, N73
R57, P69, Y201, M74, Q53, N76, D50, A56, N73
Q108, P191, Y107, R102, Q111, S192, D103, A105, N2
Epi#18
R57, L59, T61, P67, N64
R57, L59, T61, P65, N64
Epi#19
E167, N164, S192, Q108, R102, K7
E198, N194, S192, Q108, R102, K7
D103, T100, C8, Q108, R102, K7
Epi#22
L9, D103, T100, K10
A105, D103, L9, K7
D50, L45, D43, T37, K38
S147, D150, L152, K153
Epi#23
K196, N197, E199, N164, Q202, P70
K145, N197, E199, N164, Q202, P69
Epi#24
E198, K196, E199, P142, T203, P69, K143
E198, K145, E199, P142, T203, P70, K204
E198, K196, E199, P142, T203, P70, K72
E198, K145, E199, P142, F146, F151, K196
Epi#25
R57, K54, D50, N76
R57, K54, D50, E47
Epi#27
D43, E40, D125, K122
D50, E47, D43, K38
Epi#28
Q202, E199, K196, F151, S147, K144
Q202, E199, K196, F195, S147, K145
Epi#29
G58, R57, L59, R62, E136
G148, K145, L200, F195, E199
G148, K145, L200, F195, E198
Epi#33
K23, Y19, P24, S21, A16, K18
K23, Y34, P24, S21, A16, R102
Epi#34
I176, W180, T116, L115, G117, T119, S118
V31, P24, S21, L22, G35, Y34, T37
Epi#37
P69, R57, L59, K54, D50
P70, R57, L59, R62, D135
A56, R57, L59, R62, N64
P69, R57, L59, R62, D139
Epi#39
E199, L200, T203, P70, G58, L59
E198, L200, T203, P69, G58, L59
Epi#40
R57, L59, G58, T203, P69, T61
R57, L59, A56, Y201, K204, T203
R57, L59, A56, Y201, K72, T203
Epi#41
P24, Y19, L22, S21
P24, Y34, L36, S33
Epi#42
P191, S192, Q111, H98, R102, Q108
Epi#44
L59, R57, P70, Y201, A56, G58, T61
L59, R57, P69, Y201, A56, G58, T203
L59, R57, P70, Y201, A56, G58, P67
Epi#45
K153, H156, F151, Y140, N149, D150, L152
D135, H156, F151, Y140, N141, D150, L152
K143, P142, F146, Y140, N149, D150, L152
Epi#47
G58, L59, R57, M74, A56, Q202, N73, P70, P69
G148, Y140, R62, L59, R57, A56, N73, P70, P67
G66, G63, R62, L59, R57, A56, N73, P70, P67
G155, E136, R62, L59, R57, A56, N73, P70, P67
Epi#48
Q202, K204, P69, P67, G58
Q202, K204, P70, P67, G63
Q202, K72, P70, P67, G66
Epi#49
D125, D43, L45, V78, Q42, Q39, T37, K38
D125, D43, L45, V78, Q42, Q39, T37, K41
Epi#50
H98, Y96, W90, L22, S21
H98, Y96, W90, P24, S33
Epi#52
F0, A16, R102, W90, N25, Q95
F0, A16, R102, W90, N25, Q93
Betv1:
Epi#03
SAS: 270, Size 11.07: L24, K20, H76, I23, Y81
SAS: 204, Size 11.96: L24, K20, A16, I23, Y81
Epi#05
SAS: 298, Size 14.01: G110, A106, A16, P14, G111, T10
SAS: 242, Size 14.01: G110, A106, A16, P14, G111, T107
Epi#08
SAS: 464, Size 11.12: K123, E127, G1, H121, F3
SAS: 455, Size 12.95: K129, E127, G1, H121, F3
SAS: 438, Size 13.31: K123, D125, G1, H121, F3
SAS: 428, Size 11.12: K123, E127, V2, H121, F3
SAS: 425, Size 11.65: K123, E127, G124, H121, F3
Epi#09
SAS: 466, Size 20.55: D109, A106, V105, K80, A16, T77
SAS: 444, Size 20.55: D109, G110, V105, K80, A16, T77
SAS: 427, Size 20.55: D109, G111, V105, K80, A16, T77
SAS: 398, Size 19.17: T10, G110, V105, K80, A16, T77
SAS: 381, Size 19.17: T10, G111, V105, K80, A16, T77
Epi#10
SAS: 558, Size 15.18: D75, T77, N78, A106, F79, R17, K20
SAS: 549, Size 21.96: E6, T7, N4, F3, G1, K123
SAS: 517, Size 13.31: D75, T77, N78, A16, F79, R17, K20
SAS: 497, Size 15.13: D75, T77, N78, A16, F22, R17, K20
Epi#12
SAS: 335, Size 9.08: T7, Y5, E6, N4
SAS: 331, Size 11.28: R145, Y150, E148, L152
SAS: 326, Size 10.37: R70, Y83, E73, P50
SAS: 311, Size 10.32: I116, Y5, E6, N4
SAS: 308, Size 8.33: R145, Y150, E148, S149
Epi#18
SAS: 328, Size 24.67: S117, K103, F79, V105, A16, Y158, L24
Epi#22
SAS: 533, Size 9.96: D125, D93, K123, E127
SAS: 533, Size 9.96: D93, D125, K123, E127
SAS: 476, Size 11.40: D125, D93, K123, E96
SAS: 476, Size 11.40: D93, D125, K123, E96
SAS: 400, Size 17.99: D125, D93, P90, E87
Epi#23
SAS: 451, Size 22.02: K68, N43, E42, S57, F64, P63
SAS: 450, Size 22.02: K55, N43, E42, S57, F64, P63
SAS: 428, Size 22.02: K68, N43, E42, S57, L62, P63
SAS: 427, Size 22.02: K55, N43, E42, S57, L62, P63
SAS: 412, Size 18.85: K68, N43, E42, S40, F30, P35
Epi#24
SAS: 734, Size 18.92: E127, K123, E96, P90, S136, E131, K129
SAS: 729, Size 18.92: D93, K123, E96, P90, S136, E131, K129
SAS: 716, Size 19.57: E127, K123, E96, P90, S136, E131, K134
SAS: 711, Size 19.57: D93, K123, E96, P90, S136, E131, K134
SAS: 708, Size 20.49: D125, K123, E96, P90, S136, E131, K129
Epi#25
SAS: 467, Size 12.68: R70, K55, I44, E42, E45
SAS: 425, Size 12.68: R70, K54, I44, E42, E45
SAS: 420, Size 14.01: R70, K55, I44, D27, E42
Epi#27
SAS: 613, Size 14.25: D93, E127, A130, E131, K129
SAS: 595, Size 16.54: D93, E127, A130, E131, K134
SAS: 592, Size 16.70: D125, E127, A130, E131, K129
SAS: 574, Size 19.79: D125, E127, A130, E131, K134
SAS: 524, Size 18.78: D93, E127, A130, E131, K137
Epi#28
SAS: 869, Size 21.93: V33, Q36, F58, E60, L62, F64, P63, K65
SAS: 837, Size 21.83: V33, Q36, F58, E60, L62, F64, G61, K65
SAS: 808, Size 24.56: V33, Q36, F58, E60, L62, F64, P90, K65
SAS: 783, Size 21.83: V33, Q36, F58, E60, K65, F64, S57, K68
SAS: 782, Size 21.83: V33, Q36, F58, E60, L62, F64, S57, K65
Epi#29
SAS: 516, Size 9.52: G61, K65, L62, E60
SAS: 440, Size 8.7: G61, P63, L62, E60
SAS: 371, Size 6.78: G61, P59, L62, E60
Epi#32
SAS: 374, Size 17.88: F79, A16, A106, D109, V12
SAS: 354, Size 20.42: F22, A16, A106, D109, V12
Epi#33
SAS: 541, Size 18.79: K65, F64, P90, S136, A135, K134
SAS: 498, Size 9.15: Q36, F30, P35, S39, K32
SAS: 496, Size 11.27: Q36, F30, P35, S40, K32
SAS: 494, Size 12.19: Q36, F58, P35, S39, K32
SAS: 493, Size 18.79: K65, Y66, P90, S136, A135, K134
Epi#36
SAS: 447, Size 19.17: T77, A16, A106, V12, G110, T10
SAS: 430, Size 19.17: T77, A16, A106, V12, G111, T10
SAS: 392, Size 19.17: T77, A16, A106, V105, G110, T10
SAS: 391, Size 19.17: T77, A16, A106, V12, G110, T107
SAS: 375, Size 19.17: T77, A16, A106, V105, G111, T10
Epi#40
SAS: 246, Size 21.55: A106, A16, Y158, S155
SAS: 223, Size 13.25: A135, A130, Y5, T7
SAS: 196, Size 14.88: A135, A130, Y5, S117
SAS: 178, Size 10.62: A135, G140, T142, S136
Epi#44
SAS: 530, Size 19.04: L24, R17, D156, Y150, S149, V12, T10
SAS: 492, Size 19.04: I23, R17, D156, Y150, S149, V12, T10
SAS: 490, Size 17.39: L24, R17, D156, Y150, S149, V12, P14
SAS: 483, Size 23.09: L24, R17, D156, Y158, A16, A106, P108
SAS: 474, Size 20.83: L24, R17, D156, Y150, S149, V12, T107
Epi#45
SAS: 606, Size 21.41: K32, P35, F30, Y150, R145, V12
SAS: 546, Size 20.89: K32, P31, F30, Y150, R145, V12
SAS: 533, Size 15.19: K32, P35, F30, Y150, R145, G140
SAS: 533, Size 12.63: K32, P35, F30, Y150, R145, V33
SAS: 532, Size 19.60: K32, P35, F30, N28, D27, I44
Epi#47
SAS: 333, Size 21.03: R17, L24, N28, P31, P35
SAS: 300, Size 22.72: R17, L24, N28, P31, S39
SAS: 298, Size 21.80: R17, L24, N28, P31, S40
SAS: 269, Size 24.87: R17, L24, N28, P31, S57
Epi#48
SAS: 436, Size 14.26: S57, K65, P90, P63, G61
SAS: 414, Size 17.96: S39, K32, P35, P59, G61
SAS: 412, Size 17.96: S40, K32, P35, P59, G61
SAS: 389, Size 18.32: S57, K65, P63, P90, G92
SAS: 365, Size 21.15: S57, K65, P59, P35, V33
“SAS” is solvent accessible surface. “Size” is
the total suface area of the epitope in Å2.
Derf2:
Epi#02
A98, K100, S101, P99, R128, R31
A98, K100, R128, P99, R31, V94
T91, N93, P95, P34, R31, R128
L61, N93, P95, P34, R31, R128
Epi#03
L40, K15, A39, I13, Y86
L40, K14, A39, I88, Y90
Epi#05
G32, A98, R31, P34, G20, T36, T91, Y90
G32, A98, R31, P34, G20, T36, T91, V94
G32, A98, R31, P34, G20, T36, T91, L37
G32, A98, R31, P34, G20, T36, T91, V18
Epi#06
A98, P99, D129, R31, K96, P95
G32, P99, D129, R128, R31, P95
A98, P99, D129, R31, K33, P95
A98, P99, D129, R31, K96, P34
A98, P99, D129, R128, K126, P26
Epi#07
T107, S57, D59, S101, R128, A98, P99, D129
T107, S57, D59, S101, R31, A98, P99, D129
Epi#08
K15, D87, V76, H74, F75
K14, D87, V76, H74, F75
K77, D87, V76, H74, F75
Epi#09
L61, D64, I68, H74, F75, T70, N71
N114, N46, D113, K48, N71, T70, T49
G83, N46, D113, K48, N71, T70, T49
Epi#10
L40, I13, D42, N44, V81, K48, N46, N114, G115
L40, I13, D42, N44, V81, K82, N46, N114, G115
L37, D19, G20, V18, V3, D4, K6, A120, T107, V105
Epi#11
F75, K51, I111, Q45, V116, D113
F75, K51, I111, Q45, V81, D113
Epi#12
Y90, E38
Epi#13
H30, R31, P95, A98, P99, S101, G60, L61
Epi#15
K96, P99, D129, I28, R128, A98
K96, P99, D129, I127, R128, A98
K96, P99, D129, I29, R128, A98
K55, P66, D64, I68, T70, G67
Epi#18
R31, R128, I28, G125, T123, H124, V105
R31, R128, I127, G125, T123, H124, V105
Epi#22
D1, M17, D4, V3, K6
D1, M17, D19, P34, K96
D1, M17, D4, V5, K6
Epi#23
K14, N11, E12, N44, Q85, P79
K14, N11, E12, N10, Q45, P79
K14, N11, E12, N44, Q84, P79
K14, N11, E12, L40, Q85, P79
Epi#24
D129, K100, E102, P99, R128, R31, K96
E62, G60, E102, P99, R128, R31, K96
D129, K126, E102, P99, R128, R31, K33
D129, K126, E102, P99, R31, P95, K96
Epi#25
R31, K96, I97, D59, E62
R128, R31, I97, D59, E102
R128, K126, I127, E102, N103
Epi#27
D64, E62, D59, K100
D59, E62, D64, 55
D87, E38, D19, K33
D19, E38, D87, K15
D19, E38, D87, K14
D19, E38, D87, K77
Epi#28
V16, D87, Q85, K14, E12, K15, Q2, D1
I13, D87, Q85, K14, E12, K15, Q2, D1
V3, D1, Q2, K15, E12, K14, Q85, D87
L40, D87, Q85, K14, E12, K15, Q2, D1
I88, D87, Q85, K14, E12, K15, Q2, D1
V76, D87, Q85, K14, E12, K15, Q2, D1
V18, D1, Q2, K15, E12, K14, Q85, D87
Epi#29
G32, N93, L61, E62
V94, N93, L61, E62
Epi#30
G60, 197, A98, H30, K96, P34, P95
I68, N71, H74, K77, P79, V81
G32, I97, A98, H30, K96, P95, P34
Epi#34
V105, P26, S24, G125, R128, S101, P99
W92, P34, T91, V94, R31, S101, P99
I28, P26, T123, G125, R128, S101, P99
Epi#37
A120, V16, L40, K14, N11
A39, V16, L40, K14, N11
Y90, A39, L40, K14, N11
Y86, A39, L40, K14, N11
Epi#39
A120, E38, T91, P34, G20, L37
A39, E38, T91, P34, G20, L37
Epi#40
G20, L37, A120, T123, K6, S24
A39, L37, A120, T123, K6, S24
G20, L37, A120, T107, K6, T123
Epi#41
P34, L37, V106, S57
Epi#42
P26, S24, G125, R128, R31
P99, S101, G125, R128, R31
Epi#44
V16, Q2, D19, P34, W92, Y90, A39, V18, T91
V16, Q2, D19, P34, W92, Y90, A39, V5, T123
V3, Q2, D19, P34, W92, Y90, A39, V18, T91
Epi#45
K77, H74, F75, N71, D69, G67
K77, H74, F75, N71, D69, V76
K77, H74, F75, N71, D69, V65
Epi#46
A98, R128, R31, P95, N93, G32
A98, R128, R31, P34, G20, Q2
Epi#48
Q2, D19, P34, P95, G32
H30, K96, P95, P34, G20
Epi#49
D87, D42, L40, Q85, Q84, C78, T47, Q45, K48
D87, D42, L40, Q85, Q84, C78, T47, Q45, K82
Epi#50
D19, W92, P34, T91
D19, W92, P34, P95
D19, W92, T91, T36
Epi#51
D129, H30, K33, R31, R128, K126, H124
R31, H30, D129, R128, K100, K126, H124
T123, H124, K126, R128, R31, K33, H30
Derp2:
Epi#03
L17, K89, A39, I13, Y86
L17, K89, A72, I88, Y90
L17, K89, A72, I52, Y90
Epi#04
K15, S1, Q2, K14, V16, L17
K15, S1, Q2, K14, A39, L17
K15, S1, Q2, K14, V40, I13
Epi#05
G60, A56, L61, P99, G32, R31, H30, I97
G60, A56, L61, P99, G32, R31, H30, I28
Epi#06
G60, A56, D64, S57, K55, P66
G83, N46, D114, T49, K48, P79
G60, N103, D59, S101, R31, P95
Epi#08
K55, D64, S57, V106, F35
K55, E62, S57, V106, F35
Epi#09
L61, G60, E102, R128, I28, K126, N103, T123, V105
L61, G60, E102, R128, I127, K100, N103, T123, V105
L61, G60, E102, R128, I127, H124, N103, T123, V105
Epi#10
SAS: 435, Size 24.47: D69, T91, N93, F35, G32, R31
SAS: 422, Size 20.74: E38, T91, N93, F35, G32, K96
Epi#11
K14, I13, Q85, V81, E42
K15, I13, Q85, V81, E42
K14, I13, Q85, V40, D87
Epi#12
Y86, E42
Y90, E53
Y90, E38
Epi#13
H30, A125, P26, T123, A122, P19, L37, P34, W92
H30, A125, P26, T123, A122, H124, S24, G23, G20
H30, A125, P26, T123, A122, P19, L17, G20, F35
Epi#15
K55, P66, D69, I68, K89, A72
K55, P66, D69, I68, K89, A39
K55, P66, D64, I54, K109, G115
K55, P66, D64, I54, K109, A9
Epi#18
R31, I29, A125, S101, E102, N103
R31, I29, A125, S101, E102, V104
R31, I29, A125, T123, A122, V105
Epi#22
D69, P66, D64, V65, K55
D64, P66, D69, T91, K89
D59, L61, D64, P66,W92
D59, L61, D64, V65, E62
D69, P66, D64, V65, E53
Epi#24
D64, K55, E62, P99, R31, P34, K96
E53, K55, E62, P99, R31, P95, K96
D64, K55, E62, P99, R31, A98, K96
Epi#25
R31, H30, I28, E102, N103
R128, K126, I127, E102, N103
R128, K126, I28, E102, V105
Epi#27
D64, E53, D69, K89
D69, E53, D64, K55
D59, E62, D64, K55
Epi#28
V40, D87, Q85, E42, Q84, G83, K82
G20, H22, Q2, L17, E38, L37, Q36, P34, K33
G20, H22, Q2, L17, E38, L37, F35, P34, K33
Epi#29
I97, K100, L61, E62
G60, N103, L61, E62
I127, N103, L61, E62
Epi#30
G60, N103, S101, H30, K96, I97, P95
G60, N103, A125, H30, K96, I97, P95
I28, I127, A125, H30, K96, I97, P95
Epi#33
Q36, F35, V106, S57, A56, K55
K33, F35, V106, S57, A56, K55
Epi#34
I28, P26, S24, G23, G20, T123, S57
I28, P26, S24, V3, G20, T123, T107
W92, P34, T91, V18, G20, T123, P26
Epi#37
P66, V63, L61, K100, N103
P95, A98, L61, K100, N103
P19, V18, L17, K89, D87
P19, V3, L17, K89, D87
T123, V104, L61, K100, N103
Epi#38
L61, G60, E102, A125, V105, N103, P99, S57
L61, G60, E62, A56, V105, N103, P99, S57
Epi#39
A125, E102, H124, T123, P26, G20, L17
Epi#40
G60, L61, A56, T107, K6, T123
A39, L17, G20, T123, P26, S24
G60, L61, A56, T107, K55, S57
G60, L61, A56, T123, K126, S101
Epi#41
P19, L17, V3, S1
P19, L17, V5, S24
Epi#44
V65, D64, P66, W92, Y90, A39, V18, P19
L61, D64, P66, W92, Y90, A39, V18, T91
Epi#45
R31, P34, F35, N93, V94
K96, P34, F35, N93, G32
Epi#47
I127, S101, R31, I97, A98, L61, N103, P99, P95
I28, S101, R31, I97, A98, L61, N103, P99, S57
Epi#48
H30, K96, P95, P99, G60
H30, K96, P34, P19, G20
H30, K96, P34, P19, V18
H30, K96, P34, P95, V94
H30, K96, P34, P19, V3
E38, K89, P70, P66, V65
H30, K96, P95, P34, G32
Q36, K89, P70, P66, V65
Epi#50
D69, Y90, W92, P66, P70
D69, Y90, W92, P34, P95
D69, Y90, W92, T91, P34
D69, Y90, W92, V94, P95
D69, Y90, W92, L37, P19
Epi#51
K126, H124, E102, R128, I28, R31, H30
T123, H124, K126, R128, I28, R31, H30
D4, H124, K126, R128, I28, R31, H30
PhIp2:
Epi#02
T87, K85, Q61, S38, R34, R67
T87, K85, Q61, P63, R34, V42
Epi#03
K10, A90, I88, Y86
K10, A18, I88, Y86
Epi#04
R34, S38, Q61, K85, T87, I88
R34, S38, Q61, K85, T87, A90
Epi#05
G47, A18, S12, T87, G89, T91, T5, V1
G73, A29, L69, T27, G50, T53, T45, V42
G11, A18, L20, T91, G89, A90, T87, I88
Epi#06
A93, P94, D79, R34, Q61, P59
A93, P94, D79, R34, Q61, P83
A93, P94, D80, R34, Q61, P59
A93, P94, D79, R34, Q61, P63
Epi#08
K10, E9, G11, A18, H16, F54
K46, E48, G47, A18, H16, F54
K10, E9, S12, A18, H16, F54
Epi#09
L69, T27, G73, N76, R67, V77, D79, R34, A43, T45, V42
L69, T27, A29, E30, R67, V77, D80, R34, A43, T45, V42
Epi#10
D55, A18, N13, S12, F54, G47, K46
T45, A18, N13, S56, F54, G47, K46
Epi#09
L60, S56, E57, D55, K15, N13, S12, G11
L60, S56, E57, D55, H16, F54, T45, T53
L60, S56, E57, D55, H16, F54, T45, G47
Epi#12
Y86, E84
Y23, E24
Epi#18
N76, R67, F78, V81, A93, Y92, T91, T5, P2, V1
Epi#19
D39, W41, S38, Q61, R34, G37
E40, W41, S38, Q61, R34, A43
Epi#22
D79, P94, D80, P83, K85
D79, P94, D80, P63, K85
Epi#23
K10, N13, E14, L60, Q61, P59
K10, N13, E14, L60, Q61, P83
K10, N13, E14, L60, Q61, P63
Epi#24
E58, K15, E57, P59, S56, E14, Q61
D55, K15, E57, P59, S56, E58, Q61
Epi#25
R34, R67, W41, D39, E40
Epi#26
S38, E40, W41, V42, E32, E30
S38, E40, W41, V42, A43, E32
Epi#27
E14, E57, E58, K15
D55, E14, E84, K85
Epi#28
G37, H36, Q61, K85, E84, L60, F54, A43, K46
G37, H36, Q61, K85, E84, L60, F54, S12, D55
G37, H36, Q61, K85, E84, L60, F54, S56, D55
G37, H36, Q61, K85, E84, L60, F54, A43, R67
G37, H36, Q61, K15, E57, L60, F54, A43, K46
G37, H36, Q61, K85, E84, L60, F54, S12, K15
G37, H36, Q61, K85, E84, L60, F54, S56, K15
G37, H36, Q61, K85, E84, L60, F54, A43, R34
G37, H36, Q61, K85, E84, L60, F54, A18, D55
Epi#29
G73, K72, L69, R67, E30
I88, N13, L60, F54, E57
G25, K72, L69, R67, E32
V77, K75, L69, R67, E30
G37, H36, L60, F54, E57
G37, Q61, L60, F54, E57
Epi#30
I88, N13, S12, H16, K15, P59, L60
I88, N13, S56, H16, K15, L60, P59
I88, N13, A18, H16, K15, P59, L60
Epi#33
K46, F54, V42, S56, K15
H16, F54, V42, S56, K15
Epi#34
V1, P2, T5, V4, P94, Y92, T87
V1, P2, T5, L20, G89, T91, T87
V81, P94, T5, V1, P2, Y92, T91
Epi#37
T27, A29, L69, K72, D26
A43, R67, L69, K75, N76
Epi#38
L20, G89, E9, A18, N13, P59, S56
Epi#40
G49, L20, G89, Y86, K85, T87
G49, L20, G89, T87, K10, S12
G49, L20, G89, T87, K10, T7
Epi#44
V77, R67, D79, P94, Y92, A93, V1, P2
L69, R67, D79, P94, Y92, A93, V1, T5
Epi#45
D79, P94, F78, N76, M74, L69
D80, P94, F78, R67, D79, V77
K3, P94, F78, N76, M4, G73
Epi#46
A43, R67, R34, P63, H36, Q61
V77, R67, R34, P63, H36, G37
L69, R67, R34, P63, G37, Q61
Epi#47
G37, E35, E40, A43, R34, L60, N13, P59, S56
V77, E32, E40, A43, R34, L60, N13, P59, S56
S38, G37, E40, A43, R34, L60, N13, P59, S56
Epi#48
E24, K3, P94, P2, V1
E84, D80, P94, P2, V1
Epi#50
D39, W41, A43, T45
D39, W41, V42, T45
Epi#51
D79, H36, E84, T87, K10, G11, H16
D39, H36, Q61, K85, P63, R34, W41
D79, H36, E40, D39, G37, R34, W41
Q61, H36, E84, T87, K10, G11, H16

Example 11

For this example a third-generation epitope sequences were determined for some additional enzymes and redetermined for all of the enzymes in example 1-3. New enzymes are AMG (AMG pdb), BPN′ (1sup.pdb), Esperase (structure see Appendix D), Natalase (structure modelling based on SP722), Amylase-AA560 (Structure modelling based on SP722), Protease A, Alcalase, Protease B, ProteaseC, ProteaseD, ProteaseE, Properase and Relase based on their sequences and structures. The structures of Protease B, Properase, Relase, Protease A, Alcalase, ProteaseC, ProteaseD and ProteaseE can be found by “Homology modelling” (see above) and computer modelling of the epiope patterns that had been assembled in our database (shown in Table 8). Furhermore, the epitope sequences were redetermined for CAREZYME, Laccase, PD498, Savinase, Amylase SP722, and Cellulase, according to the method.

The protein surface is scanned for epitope patterns matching the given “consensus” sequence of about 6-12 residues. First, residues on the protein surface that match the first residue of the consensus sequence are identified. Within a specified distance from each of these, residues on the protein surface that match the next residue of the consensus sequence are identified. This procedure is repeated for the remaining residues of the consensus sequence. The method is further described under the paragraph “Methods” above and the program can be found in Appendixes.

The critical parameters used in this screening included:

• • i) a maximal distance between the alpha-carbon atoms of subsequent amino acids,
  • ii) a minimal accessability of the amino acid of 20 Å2,
  • iii) the largest maximal distance between the most distinct amino acids should be less than 25 Å
  • iv) the best epitope were taken,
  • v) the homology with the epitope pattern of interest was 100%

In this way a number of potential epitopes are identified. The epitopes are sorted according to total surface accessible area, and certain entries removed:

• • 1) Epitopes that contain the same protein surface residue more than once. These are artefacts generated by the described algorithm.
  • 2) Epitopes which are “too big”, i.e. where a distance between any two residues in the epitope exceeds a given threshold.

The subtilisin sequences and positions mentioned in the following are not given in the BPN′ numeration but in the subtilisins own numeration (see the alignement as described above in Tables 1A and 1B).

The epitope sequences found were:

AMG:
Epi#01
L104, P123, P107, R125, R122, N182, S184, Q172, T173
L104, P107, P123, R125, R122, N182, S184, Q172, S453
L104, P107, P123, R125, R122, N182, S184, Q172, T452
Epi#02
L234, R241, S240, F237, T173, Y175, R122, R125
L234, R241, S240, F237, T173, Y169, R125, R122
L234, R241, S240, F237, T173, Y175, R125, R54
Epi#03
L291, K404, I288, Y289
L66, K61, H254, I253, Y329
Epi#04
R122, Y175, S184, Q172, Y169, A454, I455
R122, Y175, S184, Q172, Y169, N171, A451
R125, Y175, S184, Q172, Y169, T452, A451
Epi#06
G31, A24, D25, S30, A27, P41
G146, N145, D144, T148, S149, P467
A471, N145, D144, T148, S149, P467
Epi#07
G294, T290, S405, D293, S287, R286, P307, D283
G294, T290, S287, D293, S296, R286, P307, D283
G207, T204, S200, D214, S209, R160, P157, D153
G294, T290, S405, D293, S287, R286, P307, D309
Epi#08
A27, D25, S30, V111, F49
A24, D25, S30, V111, F49
Epi#09
S149, T148, G146, N145, A471, R68, N69, T72, V470
S73, S76, T72, N69, R68, A471, N145, T148
Epi#10
D238, N182, N236, S240, F237, R241, K244
D238, T173, N182, S239, F237, R241, K244
Epi#11
F49, F109, I91, Q85, E113
Epi#12
Y363, E342
Y311, E308
Y175, E180
Epi#13
S119, W120, P123, A102, P94, S92, G90, L98
S119, W120, P123, A102, P94, S92, G96, G90
Epi#15
K244, P307, D283, I288, T290, G294
R160, P157, D153, I154, T462, G90
R286, P307, D283, I288, T290, G294
Epi#16
L410, P46, Y48, R413, S397, S394, A392, A393, N395
R160, P157, Y458, G456, S211, S209, A205, A201, D214
Epi#17
A201, S209, R160, S459
A205, S209, R160, S459
Epi#19
D44, N45, S411, Q409, R413, L410
D47, N45, S411, Q409, R413, L410
Epi#20
K61, P434, L66, L423, N427, D65, G70, D71
Epi#22
D357, S356, D349, V346, D345
D349, S356, D357, A359, D345
D357, S356, D349, L348, D345
Epi#23
K404, N292, E299, S298, L295, A300
K404, N292, E299, S296, L295, A300
Epi#24
D336, K337, E259, P258, S431, L332, K378
D336, K337, E259, P258, S431, R429, K378
D336, K337, A261, P258, S436, E259, Q338
Epi#25
R125, R122, W120, E180, N182
R241, K244, E308, N313
Epi#26
W212, S200, E198, W437, V197, G438, E259
W212, S200, E198, W437, V197, A201, D214
Epi#27
D283, E280, D349, K352
D403, E408, D406, K404
D349, E280, D283, K244
D349, E280, D283, K279
Epi#28
L332, D336, Q338, K337, E259, C262, P272, D345
V374, D336, Q338, K337, E259, C262, P272, D345
G339, D336, Q338, K337, E259, C262, P272, D345
Epi#29
L295, G294, L291, R286, E299
I288, K404, L291, R286, E299
L348, K352, L354, F380, E299
Epi#33
K352, Y355, V374, S371, S365, K337
K352, Y355, V374, S365, S340, K337
Epi#34
V463, W466, S468, V470, P467, T464, T462
I469, W466, S468, V470, P467, T464, T462
I154, W466, S468, V470, P467, T464, T462
V463, W466, S468, V470, P467, S465, T464
Epi#37
T362, A359, L348, K352, D357
T360, V346, L348, K352, D357
T362, A359, L348, K352, D349
Epi#38
G438, E259, A435, R68, L66, N69, P434, S431
Epi#39
A353, E299, R286, P307, G243, L234
A300, E299, R286, P307, G243, L234
Epi#40
A205, L143, G146, Y147, P467, T464
G146, L143, A205, T204, A201, S209
A451, A450, T448, P446, S444
Epi#41
P467, Y147, L143, V206, S149
Epi#42
L66, P434, S431, N430, R429, R428
L104, P123, S95, G101, P94, R122, R125
L104, P107, S95, G96, P123, R125, Q172
Epi#44
L143, Q140, D144, W141, Y147, S468, V470, T72
V206, Q140, D144, W141, Y147, S468, V470, P467
S211, Q216, D214, P218, Y223, A451, A450, T448
S211, Q216, D214, P218, Y223, A450, G447, T448
Epi#45
R413, P46, F49, Y50, N110, D112, G31
R413, P41, F49, Y50, N110, D33, G31
D44, P46, F49, Y50, N110, D112, G31
Epi#46
Y175, R125, R122, P123, G174, Q172
Y169, R125, R122, P123, G174, Q172
V432, R429, R428, P434, N69, G70
Y175, R125, R122, P94, N93, G90
Y175, R122, R125, P123, N182, G121
Y175, R125, R122, P94, G101, A102
Y175, R125, R122, P94, G118, A115
Y175, R125, R122, P94, G101, G96
Y175, R122, R125, P123, N182, G183
Epi#48
S211, D214, P218, P446, G447
E259, K337, P258, P434, V432
S215, D214, P218, P446, G447
S209, D214, P218, P446, V445
E259, K337, P258, P434, V433
Epi#50
R122, Y175, W120, T117, S119
R125, Y175, W120, S119, T117
Epi#51
T390, H391, E408, Q409, R413, S411, W317
T390, H391, E408, S405, I288, K404, W317
D406, H391, E408, Q409, R413, S411, W317
T390, H391, E408, D406, K404, Q409, W317
Epi#52
W437, A260, T266, R273, W228, D264, Q225
BPN′:
Epi#02
T255, K256, S260, F261, P194, Y262, R186, V203
L257, K256, S260, F261, P194, Y262, R186, V203
T253, K256, S260, F261, P194, Y262, R186, V203
Epi#03
K141, A137, I108, Y104
K136, A137, I108, Y104
K136, A134, I108, Y104
Epi#04
K265, Y262, S188, Q185, R186, N184, L257
k265, Y262, S188, Q185, Y263, R186, L257
K265, Y262, S188, Q185, R186, N184, G258
K265, Y262, S188, Q185, Y263, R186, G258
Epi#05
G80, A1, N77, P40, G211, S38, S37, V44
G80, A1, N77, P40, G211, S38, S37, L42
G127, A152, N155, T164, G160, S158, S188, Y262
Epi#06
G211, N212, D36, S37, K43, P40
G80, N212, D36, S38, K43, P40
G211, N212, D36, S38, K43, P86
Epi#08
K256, D259, S260, F261
K43, D36, S38, V44, F58
Epi#09
S105, S132, A133, A137, D140, K141, A144, S145, N118
S248, T244, A144, S145, D120, K27, N118, A116, N117
Epi#10
E54, T55, N57, S37, F58, G46, K43
T55, A48, N57, S37, F58, G46, K43
E54, T55, N57, S49, F58, G46, K43
Epi#11
K136, I108, Q103, V51, D98
Epi#12
Y171, E195
Epi#13
S101, W106, P52, T55, A48, P56, S49, G47, F58
S105, W106, P52, T55, A48, P56, S49, G47, W113
Epi#15
N25, P239, D120, I115, K141, A144
N240, P239, D120, I115, K141, A144
Epi#16
Q271, P14, Y21, G20, Q19, S18, A15, A272, N252
Q59, P210, Y214, G211, S38, D36, D61, A99, D98
Epi#17
A187, S188, R186, S183
A187, S188, R186, S182
Epi#18
N184, R186, S188, G157, S158, T159, S161
N184, R186, S188, G157, S158, T159, S162
N184, R186, S188, G157, S158, E156, N155
N184, R186, S188, G157, S158, E156, F189
Epi19
E156, N155, S188, Q185, R186, L257
E156, N155, S188, Q185, R186, G258
E156, N155, S188, Q185, R186, A187
Epi#22
D197, S260, D259, L257, K256
D197, S260, D259, Y263, K256
Epi#23
N155, E156, S188, Q185, A187
Epi#24
E156, G166, E195, P194, S260, L257, K256
D259, G264, E195, P194, S260, L257, K256
D197, K170, E195, P194, S260, L257, K256
Epi#25
K141, I115, D120, N25
K141, I115, D120, N118
K141, I115, E112, N118
Epi#26
W113, S49, W106, P52, E54, D98
W113, S49, W106, P52, E54, D60
W113, S49, W106, V51, E54, D98
Epi#28
A99, D61, Q59, F58, E54, L96, Q103, G102, D98
A99, D98, Q59, F58, E54, L96, Q103, G100, D61
A99, D61, Q59, F58, E54, L96, Q103, S101, D98
Epi#29
G102, Q103, L96, E54
G100, Q103, L96, E54
Epi#30
I79, N76, S87, H17, S18, P14, V4
I79, N76, S87, H17, Q19, P14, V4
Epi#31
L257, Q185, N184, R186, F189, V203, I205, D181
L267, Q10, N184, R186, F189, V203, I205, D181
Epi#33
K213, Y214, P210, S38, S37, K43
Q59, F58, V44, S38, S37, K43
Epi#34
W106, P52, M50, G47, P56, T55, S53
W106, P52, S49, G47, P56, T55, S53
I115, W113, M50, V51, P52, T55, S53
I108, W106, S105, V51, P52, T55, S53
Epi#35
A99, L96, S49, M50, I108
A99, L96, S49, M50, I107
Epi#36
A137, A134, A133, G131, Y104, S105, Q103, V51, A48, W113
A134, A137, A133, G131, Y104, S101, Q103, V51, A48, W113
Epi#37
Y262, R186, L257, K256, D259
Y263, R186, L257, K256, N252
Epi#39
E156, T164, P129, G127, L126
E156, T164, P129, G128, L126
E156, T164, P129, G154, L126
E156, T164, P129, G166, L126
Epi#40
R247, L250, A272, T255, K256, S260
R186, L257, G258, Y263, K256, S260
G264, L257, G258, T255, K256, S260
Epi#41
P194, Y262, L257, S260
P194, Y263, L257, S260
Epi#42
P194, S260, G258, R186, Q185
Epi#44
S182, Q185, D181, Y6, S9, V4, P14
S183, Q185, D181, Y6, S3, V4, P5
S248, R247, D197, P194, Y262, S260, G258, T255
S53, P52, W106, Y104, S105, V51, T55
Epi#45
K170, P194, F261, Y262, R186, D181, V203
D197, P194, F261, Y262, R186, D181, V203
Epi#46
S162, S158, E156, N155, A187, Q185, N184, R186, S188
S188, S158, E156, N155, A187, Q185, N184, R186, S183
S158, S188, E156, N155, A187, Q185, N184, R186, S182
S161, S158, E156, N155, A187, Q185, N184, R186, S183
G160, S158, E156, N155, A187, Q185, N184, R186, S188
Epi#48
S38, K43, P40, P210, G211
S37, K43, P86, P14, V4
S38, K43, P40, P210, G215
Epi#50
H238, W241, T242, P239
H238, W241, T244, T242
H238, W241, T242, T244
Epi#51
T242, H238, Q275, Q271, P14, S18, H17
Q245, H238, Q275, K237, P239, T242, W241
Q275, H238, Q245, T242, R247, T244, W241
Q245, H238, Q275, Q271, P14, Q19, H17
CAREZYME Core:
Epi#01
P61, P165, K164, R158, N154, Y168, R153, S151
P137, P49, K44, K13, N32, Y54, Q36, T39
P61, P165, K164, R158, N154, S152, R153, S151
Epi#02
L115, N118, S117, R4, T6, Y147, R146, V129
L115, N118, S5, R4, T6, Y147, R146, V129
Epi#03
K44, A43, I38, Y54
K13, A43, I38, Y54
Epi#04
R153, S151, Q145, Y147, R146, I131
R153, S151, Q145, Y147, R146, G144
R153, S151, Q145, Y147, R146, L142
Epi#05
G3, A1, S183, T95, G101, A100, S96, G97
G3, A1, F184, T93, G101, T95, S96, G97
G97, A100, S96, T95, G101, T93, S183, G3
Epi#06
G140, P160, D161, R158, K164, P165
G50, P137, D133, R146, Q145, P143
A162, P165, D161, R158, K164, P160
Epi#07
G148, T6, S181, D178, R170, P165, D58
G128, T6, S181, D178, R170, P165, D58
Epi#08
K44, D42, S45, A43, F41
Epi#09
A191, E192, R196, A195, R200, N25, N202, N206
D161, R158, D157, R153, N176, S151, N154
Epi#10
D161, A57, N34, A162, F159, R158, K164
D2, A1, R185, S183, F184, G3, R4
Epi#11
F41, F29, I38, Q36, D58
Epi#12
Y168, E155
Y90, E91
Epi#13
A63, W62, P165, T60, A162, P160, L142, G149, Y147
A63, W62, P165, T60, A162, P160, L142, G128, Y147
A63, W169, P165, T60, A162, P160, L142, G144, Y147
Epi#15
P137, D133, I131, R146, G144
P137, D133, I131, R146, G148
P137, D133, I131, R146, G130
P137, D133, I131, R146, G128
P137, D133, I131, R146, G149
Epi#16
Q138, P137, Y54, R37, Q36, N34, A162, A57, D161
R170, P165, Y168, R153, S151, N176, D172, A63, D67
R170, P165, Y168, R153, S151, N176, D172, A63, D66
Epi#17
A1, S183, R4, S117
A100, S181, R4, S183
A1, S183, R4, S5
Epi#18
N118, R4, S181, —, G3, —, S117, L115, —, A78, S80
N34, N32, R37, F35, —, A33, Y54, S45, —, —, A43, V52
Epi#19
D157, N154, S151, Q145, R146, L142
D178, N176, S151, Q145, R146, G144
Epi#22
D40, A43, D42, W18, K20
D40, A43, D42, A19, K20
Epi#23
R158, N154, E155, L142, Q145, P143
R153, N154, E155, S151, Q145, P143
Epi#24
D42, K44, E48, P137, F139, A33, Q36
D40, K44, E48, P137, F139, A33, Q36
D161, K164, A162, P160, R158, L142, Q145
D161, K164, E155, P143, R158, L142, Q145
Epi#25
R158, K164, W169, D172, N176
R4, H119, I77, E82, N81
Epi#26
W18, S15, E82, W85, P23, A19, D42
W18, S15, E82, W85, P23, G84, D203
Epi#28
I131, D133, Q138, L142, E155, K164, F159, P165, D161
I131, D133, Q138, L142, E155, K164, F159, P143, R158
I131, D133, Q138, L142, E155, K164, F159, P160, R158
Epi#29
I131, R146, L142, R158, E155
G144, Q145, L142, R158, E155
Epi#30
G79, N81, A78, H119, S117, I77, L115
G79, N81, A78, H119, S76, I77, L115
Epi#31
L142, R158, N154, R153, W169, F171, D172
Epi#33
Q36, F29, P27, S15, A19, K20
K44, F41, P27, S15, A19, K20
Epi#34
V129, P143, S151, G144, R146, Y147, T6
V129, P143, S151, G148, R146, Y147, T6
V129, P143, S151, G149, R146, Y147, T6
Epi#36
A83, A22, A19, S15, K13, V52, A43, W18
Epi#37
Y147, R146, L142, R158, D161
Y147, R146, L142, R158, N154
Y147, R146, L142, R158, D157
Epi#38
E155, R158, P160, G140, L142
E155, R158, P143, G144, L142
Epi#40
G79, L115, G113, T111, A74, T6
G79, L115, G113, T111, A74, S15
G79, L115, G113, T111, A74, S110
G116, L115, G113, T111, A74, T6
G79, L115, G113, T111, A74, S76
Epi#42
L142, P143, S151, G144, R146, Q145
L142, P143, S151, G148, R146, Q145
L142, P143, S151, G149, R146, Q145
Epi#44
L142, R158, D161, P165, W62, Y168, S152, G144, P143
I131, R146, D133, P137, Y54, A33, V52, P49
L142, R158, D161, P165, W62, Y168, S152, G149, P143
Epi#45
R185, P208, F207, N206, D203, V24
D67, P213, F68, N65, D66, V64
R185, P208, F207, N206, D204, G205
Epi#46
A195, R200, R201, P23, N202, G205
A191, R200, R201, P23, N202, G205
V24, R201, R200, P190, Q211, A209
Epi#47
A191, A195, E192, V194, R200, N202, R201, P23
A195, A191, E192, V194, R200, N25, R201, P23
A191, A195, R196, V194, R200, N202, R201, P23
Epi#48
E48, K44, P49, P137, V52
E48, K44, P49, P137, G50
E48, K44, P49, P137, G140
Epi#50
D172, Y168, W62, V64, P213
D42, W18, A43, T39
D67, W173, W62, V64, P213
D66, W173, W62, V64, P213
D42, W18, S45, P49
D172, W169, W62, V64, P213
Epi#51
R4, H119, D2, T95, P98, K175, W169
R4, H119, D2, R185, P208, Q186, W85
R4, H119, D2, T95, G97, K175, W173
Epi#52
W18, A22, R200, R201, W85, Q186
Esperase:
Epi#01
N24, P239, R237, K235, N243, S240, Q245, T242
N24, P239, K235, R27, N117, Y91, R43, S87
N24, P239, R237, K235, N243, Y241, Q245, S240
Epi#02
T3, N76, L75, R43, S38, Y209, R213, V215
T3, N76, S87, R43, S38, Y209, R213, V215
T129, N166, Q161, R160, T156, Y192, R186, V203
Epi#03
R186, Y192, S261, Q161, R160, N155, G127
R186, Y192, S261, Q161, R160, N155, G157
R186, Y192, S261, Q161, R160, N155, L126
R186, Y192, S261, Q161, R160, T156, G162
R186, Y192, S261, Q161, R160, N155, A187
Epi#05
G102, A105, S133, T134, G131, R170, T129, Y167
G102, A105, S133, T134, G131, R170, T129, G127
G211, A37, R43, P40, G80, T3, S78, I79
Epi#06
G211, N61, D97, R98, S53, P55
G102, N99, D97, R98, S53, P55
G100, N99, D97, R98, S53, P55
Epi#07
211, T210, D60, S38, R43, P86, D89
Epi#08
A108, E136, S133, A105, F50
A108, E136, S132, A105, F50
A187, D181, S188, V203, F189
Epi#09
N212, G211, S38, H59, N61, N99, R98
S52, S53, R98, N99, N61, G211
Epi#10
T129, T156, N155, S188, F189, G157, R160
D181, N183, R186, S188, F189, G157, R160
T129, N166, N155, S188, F189, G157, R160
T129, T156, N155, S218, F189, G157, R160
D97, N99, N61, S57, F50, G102, R98
Epi#12
Y167, E136
Y192, E195
Y171, E136
Epi#13
S38, R43, P40, A37, H59, S57, P55, Y58
S38, R43, P40, A37, H59, S57, P55, F50
S38, R43, P40, A37, H59, S49, P55, Y58
Epi#15
N24, P86, D89, I44, R43, A45
N24, P86, D89, I44, R43, G46
N76, P86, D89, I44, R43, A45
N24, P86, D89, I44, R43, A37
Epi#16
Q161, P194, Y192, G157, R160, S188, D181, A187, N183
Q161, P194, Y192, R186, Q185, S188, D181, A187, N183
Q161, P194, Y192, G162, R160, S188, D181, A187, N155
Epi#17
A37, S38, R43, S87
Epi#18
N144, N140, R141, L137, S133, T134, E136, S132
N140, N144, R141, L137, S133, T134, A105, S103
N143, N144, R141, L137, S133, T134, E136, N140
Epi#19
I21, N18, Q15, Q275, R19, G20
I21, N18, Q15, Q275, R237, G20
E197, N265, S261, Q161, R160, G162
E197, N265, S261, Q161, R160, G157
I21, N18, Q15, Q275, R237, G25
Epi#23
R98, N61, E54, S53, F50, P55
R98, N61, E54, Y58, F50, P55
R98, N61, E54, S57, F50, P55
R98, N61, E54, S52, F50, A105
Epi#24
E195, G264, E197, P260, S261, P194, Q161
D89, G46, A48, P55, S52, F50, Q109
E197, G264, E195, P194, S261, L262, Q161
Epi#25
R98, H59, E54, N61
R98, H59, D60, N61
R43, H39, I44, D89, N24
R27, H120, I115, E112, N116
Epi#28
L104, Q109, I115, E112, W113, F50, S53, R98
A105, Q109, I115, E112, W113, F50, G102, R98
A108, Q109, I115, E112, W113, F50, S53, R98
V107, Q109, I115, E112, W113, F50, S53, R98
Epi#29
I147, N140, L137, R141, E136
G146, N140, L137, R141, E112
I115, N143, L137, R141, E136
G102, N99, L96, R98, E54
Epi#30
G211, N212, S38, H59, S57, I51, P55
G211, N61, S57, H59, S38, P40, L75
G211, N212, S38, H59, S49, I51, P55
G211, N212, S38, H59, P55, I51, L96
Epi#31
L257, Q185, N183, R186, F189, V203, D181
L262, Q185, N183, R186, F189, V203, D181
Epi#33
H59, Y58, P55, S52, S53, R98
Q109, F50, P55, S57, S53, R98
Q109, F50, P55, S49, S53, R98
Epi#34
I79, P40, S38, G211, R213, Y209, S216
I79, P40, S38, G211, R213, Y214, T210
I51, P55, S49, L96, R98, S53, S52
Epi#37
T134, A108, L137, R141, N144
Y256, A254, L257, R186, N183
A105, A108, L137, R141, N144
Epi#38
L257, G264, E195, L262, N265, P260, S259
L257, G264, E195, L262, N265, P260, S261
Epi#39
E195, R170, P194, G264, L257
E195, R170, P194, G264, L262
Epi#40
R141, L137, A108, T134, A105, S133
R43, L42, A37, Y58, P55, S52
R186, L257, A254, Y256, P260, S259
R186, L262, G258, Y256, P260, S259
R186, L257, G184, Y256, P260, S259
R141, L137, A108, T134, A105, S103
R186, L262, G264, Y256, P260, S259
R186, L257, A254, Y256, P260, S261
R186, L262, G258, Y256, P260, S261
R186, L257, G264, Y256, P260, S261
Epi#41
P260, Y256, L257, S259
Epi#42
L75, P86, S87, N24, P239, R237, Q275
L75, P86, S87, N24, P239, R237, R19
Epi#44
S53, R98, D97, Y58, S57, A48, P55
S53, R98, D97, Y58, S38, G211, T210
Epi#45
R19, H17, F22, N24, D89, G25
R43, P86, F22, N24, D89, G25
R272, H269, F10, N183, D181, V203
R272, H269, F10, N183, D181, G184
R43, P86, F22, N24, D89, G46
Epi#46
R19, R237, P239, N24, G20
R19, R237, P239, N24, G25
Epi#47
G162, Y192, R160, N155, A187, Q185, N183, R186, S188
G157, Y192, R160, N155, A187, Q185, N183, R186, S188
S261, Y192, R160, N155, A187, Q182, N183, R186, S188
L262, Y192, R160, N155, A187, Q182, N183, R186, S188
Epi#48
S261, Q161, P194, P260, G258
S261, Q161, P194, P260, G264
Epi#50
D181, W6, V4, T3
D181, W6, V203, S188
D181, W6, V4, S9
D181, W6, T3, P5
Epi#51
R98, H64, T210, R213, P40, S38, H59
R98, H64, T210, R213, G211, S38, H59
R19, H17, Q15, Q275, R272, Q252, H269
Laccase:
Epi#02
A14, N15, S17, F21, P180, Y176, R266, V177
T22, N15, P18, F21, P180, Y176, R266, V177
A274, N275, A181, R175, P180, Y176, R266, V177
A24, N15, S17, F21, P180, Y176, R266, V177
T272, N275, A181, R175, P180, Y176, R266, V177
Epi#03
L184, K173, I186, Y256
Epi#04
R234, S211, Q261, K264, N267, G271
R234, S211, Q261, K264, R266, G268,
R259, S211, Q302, R234, N299, A301
R259, S211, Q236, R234, N299, A301
Epi#05
G372, A371, L369, P350, G81, S349, S351, V352
G372, A371, L369, P350, G81, S351, S349, Y347
Epi#06
G286, N289, D291, T293, S295, P292
G214, P252, D254, T293, S295, P298
A288, N289, D291, T293, S295, P292
Epi#07
G214, T294, D291, R283, V253, P252, D254
G30, T12, D53, R59, A497, P89, D51
G30, T10, D51, R59, A497, P55, D53
Epi#08
A371, E348, S349, A346, F335
A14, D53, G90, A92, H91, F93
A181, E183, G20, V16, F21
A181, E183, G20, A182, F21
Epi#09
N41, A100, N43, V6, D42, R37, N4, T8, L94
N41, A100, N43, V6, D42, R37, N4, T8, N47
L369, N366, E376, R379, N472, A471, V474
Epi#10
E183, A181, N275, T272, F273, G268, R266
D129, N41, N43, A100, F69, G72, R71
E183, A181, N275, A274, F273, G271, K264
Epi#11
F93, L486, I489, Q485, V481, E482
Epi#12
Y490, E488
Y375, E376
Epi#13
N366, P370, D367, I358, Q363, A471
N366, P370, D367, I358, Q363, G361
R379, P378, D326, I319, T321, G323
R379, P378, D326, I319, T321, G318
R379, P378, D326, I319, T321, A324
Epi#15
N366, P370, D367, I358, Q363, A471
N366, P370, D367, I358, Q363, G361
R379, P378, D326, I319, T321, G323
R379, P378, D326, I319, T321, G318
R379, P378, D326, I319, T321, A324
Epi#16
R175, P180, Y176, R266, Q164, N267, D166, A163, D205
R283, P292, Y256, G214, Q251, D254, A285, A288, N289
R283, P292, Y256, G214, Q251, D254, D291, A290, N289
Epi#17
A306, S413, R409, S414
A411, S413, R409, S414
A306, S410, R409, S414
A411, S414, R409, S410
Epi#19
E216, N250, Q251, Q191, R283, G286
E190, N250, Q251, Q191, R283, A288
E216, N250, Q251, Q191, R283, A290
E190, N250, Q251, Q191, R283, A285
Epi#22
D491, P494, D492, P495, E496
D492, P494, D491, L493, E496
Epi#23
R339, N460, E348, S349, L369, A371
R339, N460, E348, S351, L369, P370
R339, N460, E348, S351, L369, A365
R339, N460, E348, S351, L369, P350
R283, N188, E190, N250, Q191, P252
Epi#24
D475, G72, A476, P445, R379, A471, Q363
D53, G90, A497, P495, T498, P55, Q501
D53, G90, A497, P495, S499, L58, Q501
Epi#25
R37, K40, D129, N130
R37, K40, D129, N41
Epi#27
E142, E139, D138, K194,
E142, E139, D138, K193
Epi#28
L58, Q501, I500, E496, L493, P495, D492
G286, D254, Q191, K194, E190, K193, G192, D138
A288, D254, Q191, K193, E190, K194, G192, D138
G192, D248, Q191, K194, E139, L136, A135, D138
V253, D254, Q191, K193, E190, K194, G192, D138
A285, D254, Q191, K193, E190, K194, G192, D138
Epi#29
G390, Q332, L329, R330, E435
V374, N366, L369, E348
I500, P495, L493, E496
G344, Q332, L333, R330, E435
Epi#30
G412, N304, A306, H309, I312, P314, V419
I312, L311, A315, H309, P229, L136, P132
Epi#31
L329, Q332, N343, R330, F331, V386, D434
L333, Q332, N343, R330, F331, V386, D434
L58, Q501, N54, R59, F112, M459, F456, D205
L58, Q501, N54, R59, F112, M459, I454, D205
Epi#33
Q485, Y490, P494, S499, A497, R59
Q251, Y256, P292, S295, A296, R234
H153, F21, V16, S17, A182, K173
H153, F21, P18, S17, A182, K173
Epi#34
V431, P395, T432, G433, G412, T415, S414
V431, P388, T432, G412, G433, S414, T415
V419, P320, T321, G323, P322, Y416, S414
V431, P395, T432, G390, G433, S414, T415
Epi#35
A371, L369, A362, S360, M359, I358
G372, L369, A362, S360, M359, I358
A365, L369, A362, S360, M359, I358
Epi#36
A362, A471, A476, V474, G361, S360, Q357, P350, A371, A365
A290, A288, A285, V253, Y256, S295, A296, W257
A288, A285, A287, V253, Y256, S295, A296, W257
Epi#37
P132, A135, L136, K194, N250
A135, A134, L136, K194, D138
P298, A301, L303, R234, N299
Epi#38
L356, G81, E348, A371, V374, L369, N366, P370, S351
L356, G81, E348, A371, V374, L369, N366, P370, S349
Epi#39
A411, E435, T432, P395, G393, L392
A1, E142, L35, R37, P34, G30, L27
A389, E435, T432, P395, G394, L392
Epi#40
R330, L333, G390, T432, A411, S414
G393, L392, G394, T432, A411, S414
R330, L333, G390, T432, A411, T415
Epi#41
P370, L369, V352, S351
P350, L369, V352, S351
Epi#42
L392, P395, S428, G430, P388, R330, Q332
Epi#44
S360, Q363, D367, P370, Y347, A371, G372, T345
V253, Q191, D254, P292, W257, Y256, S295, A296, P298
S360, Q363, D367, P370, Y347, S349, V352, P350
V253, Q191, D254, P292, W257, Y256, S295, G214, P252
Epi#45
R409, P322, F418, Y416, N420, D313, V419
K423, P314, F418, Y416, N420, D313, V419
R175, P180, F21, Y176, R266, D166, G268
Epi#46
A296, R259, R234, P300, N299, A301
Y256, R259, R234, P300, N299, Q302
Epi#47
I212, S211, R234, L303, A301, N299, P300, P298
I212, S211, R234, V232, A301, N299, P300, P298
Epi#48
S158, Q160, P157, P155, V504
S499, Q501, P55, P155, V504
E488, Q485, P480, P479, V481
Epi#49
D367, L369, V352, P350, Q357, Q363, M359, N478
D367, L369, P370, P350, Q357, Q363, M359, N478
Epi#50
D291, Y256, W257, S295, P298
D254, Y256, W257, T293, S295
Epi#51
D307, H309, E228, T218, P229, T231, H230
R234, H215, E216, T231, P229, H230, H309
D248, H215, E216, T231, P229, H230, H309
Epi#52
F69, A100, T98, R71, W75, T73, Q70
F97, A100, T98, R71, W75, T73, Q70
Natalase:
Epi#01
P344, P382, R387, R33, N32, S28, R31, T36
P344, P382, R387, R33, N29, S28, R31, T36
Epi#02
A87, N21, Q18, R24, S28, R31, R33
A87, K89, S83, R24, S28, R31, R33
Epi#03
L307, K305, H402, I404, Y398
L307, K305, H401, I404, Y398
L307, K305, A304, I404, Y398
Epi#04
R167, S166, Q168, R172, N171, I173
R177, Y131, S128, Q125, R123, N124, I127
Epi#05
G178, A180, N124, P120, G190, S187, H234, L195
G178, A180, N124, P120, G190, R123, S187, Y192
G178, A180, N124, P120, G190, S187, H234, Y192
Epi#06
A87, N21, D25, R24, Q18, P14
G145, N146, D150, T147, R144, P142
G143, N146, D150, T147, R144, P142
G450, N451, D447, T455, K452, P453
A87, N21, D25, R22, Q18, P14
G454, N451, D447, T455, K452, P453
A378, P382, D447, T455, K452, P453
Epi#07
G145, T147, D150, S149, R213, V208, P205, D201
Epi#08
K305, D400, A304, H402, F399
K305, D400, A304, H401, F399
Epi#09
S79, S83, D25, R22, R24, H86, N90, S28, R31
N439, A460, N459, V444, K478, N417, T413, T414
Epi#10
E254, N249, R248, T245, F239, R212, R213
E254, N249, R248, T245, F239, R241, K275
Epi#11
F169, I173, Q170, D162
L195, I173, Q170, D162
Epi#12
Y192, E188
Y357, E354
Epi#13
H12, L13, P369, A375, P374, S372, P330, W11
H12, L13, P369, A375, P374, S372, P330, L334
H12, L13, P369, A375, P374, S372, P330, G331
Epi#15
N451, P453, D447, I448, T449, A378
N451, P453, D447, I448, K452, G450
Epi#16
Q313, P316, Y357, R353, Q395, D397, D400, A304, N308
Q355, P316, Y357, G356, R353, D397, D400, A304, D302
Epi#17
A87, S83, R24, S28
A87, S28, R24, S83
Epi#18
R33, N32, R31, S28, G92, N90
Epi#19
D16, N50, S48, Q49, R72, G69
D25, N21, Q80, Q18, R24, A87
E82, T77, Q18, Q80, R72, G69
Epi#22
D461, A460, W463, W433
Epi#23
K478, N417, E410, N439, Q438, A460
K478, N417, E410, N439, Q438, A441
Epi#24
E332, G331, E335, P330, S372, A375, K379
D381, K379, A375, P369, S372, P374, K377
Epi#25
R154, K138, W136, D162, N171
R213, R212, W217, E216, N249
R154, K138, W136, E134, N112
R241, K236, W183, D203, E206
Epi#26
W163, S166, E134, W136, V161, E117, E126
W163, S166, E134, W136, V161, E117, D130
W163, S166, E134, W136, V161, E117, D162
Epi#27
D203, E206, D201, K236
E117, E126, D130, K175
D201, E206, D203, K179
E126, E117, D162, K175
Epi#28
L195, D162, Q168, W163, E134, W136, Q165, S166, R167
I173, D162, Q170, W163, E134, W136, Q165, S166, R167
V161, D162, Q170, W163, E134, W136, Q165, S166, R167
Epi#29
G331, P330, L334, F337, E335
G178, K175, L114, R177, E117
Epi#30
G450, N451, H446, K478, I448, P453
G454, N451, H446, K478, I448, P453
Epi#31
Q168, N171, R172, W163, M196, I173, D162
Q170, N171, R172, W163, V161, I173, D162
Epi#33
K377, Y366, P369, S372, A375, K379
K377, Y366, P374, S372, A375, K379
Epi#34
W433, W463, T457, V444, G454, T455, P453
W433, W463, T457, V456, G454, T455, P453
Epi#37
Y156, R177, L114, K175, D130
T132, R177, L114, K175, N124
Epi#38
G429, E431, N469, P428, S472
G430, E431, N469, P428, S472
Epi#39
E10, H12, T370, P330, G331, L334
E10, L13, T370, P330, G331, L334
Epi#40
A378, A375, Y366, P369, S372
R177, L114, G178, Y156, K138, T110
A375, A378, Y366, P369, T370
Epi#41
P369, L13, V52, S48
Epi#42
P316, S281, G356, R353, Q355
P316, S281, G356, R353, Q395
Epi#44
V208, R213, W217, Y148, S149, G145, P142
S28, R33, D381, Y365, A378, A375, P369
L13, D16, P14, W11, Y362, A375, V373, T370
S333, D327, P330, W11, Y362, A375, V373, P369
Epi#45
D108, P142, F65, Y60, N146, D150, G145
D140, P142, F65, Y60, N146, D150, G145
Epi#46
Y392, R387, R33, P382, G450, G454
Y392, R387, R33, P382, Q388, G3
Epi#47
S83, S79, E82, I85, R24, A87, N90, R31, S28
A250, G252, E254, N249, R248, F256, N279, R241, S238
Epi#48
S372, H371, P374, P369, V373
Epi#49
D51, W11, L13, V52, P14, Q18, Q80, T77, N21
D51, W11, L13, V52, P14, Q18, Q80, T77, K74
Epi#50
D461, Y435, W433, W463, T457
D400, Y398, W433, W463, T457
D397, Y435, W433, W463, T457
Epi#51
T394, H396, D397, D400, K305, H402, H401
T455, H446, K478, T457, G442, Q438, W463
Epi#52
W136, A109, E134, R167, W163, N171, Q170
W136, A109, E134, R167, W163, N171, Q168
PD498:
Epi#02
T262, K258, S260, F266, T198, Y196, R168, V166
T262, K258, S260, F266, T264, Y196, R168, V166
T141, N139, Q171, F170, S167, Y196, R168, V166
Epi#03
L99, K51, A49, I53, Y56
L99, K51, A49, I53, Y43
Epi#04
R28, S331, Q333, K97, R50, I53
R28, S331, Q333, K97, R50, A49
Epi#05
G108, A106, N107, G110, S109, S111, I59
G110, A106, N107, G108, S109, S111, L112
G108, A106, N107, G110, S111, S117, Y121
G108, A106, N107, G110, S111, S109, G135
G110, A106, L68, P214, G217, S219, Y220
G108, A106, N107, G110, S111, S109, L134
Epi#06
G135, N163, D164, R168, S174, P176
G162, N165, D164, R168, S174, P176
A22, N274, D25, S2, S9, P6
G154, N152, D148, T142, K144, P176
A22, P21, D25, S2, S9, P6
G154, N152, D148, S145, K144, P176
Epi#07
29, T332, S331, D95, S240, R28, V26, P21, D25
G29, T332, S330, D95, S331, R28, V26, P21, D25
Epi#08
K258, D257, S260, F266
K190, D185, S192, V207, F193
Epi#09
N215, N44, R50, I53, K54, N64, N63, R61
N44, A49, R50, I53, K54, N63, N64, R61
Epi#10
D188, N187, R189, S260, F266, G263, K258
D185, N187, R189, S260, F266, G263, K258
Epi#12
Y268, E253
Epi#15
R50, P46, D82, I87, T83, G86
N215, P46, D82, I87, T83, G86
Epi#18
N216, N44, R50, I53, A49, P46, N215
N215, N44, R50, I53, A49, P46, N216
Epi#19
D95, T332, S240, Q241, R28, G29
D95, T332, S330, Q241, R28, G29
Epi#22
D185, S192, D164, Y196, K267
D105, S111, D113, T141, K144
Epi#24
D95, K51, A49, P46, R50, K97
Epi#25
R120, K153, W151, D148, N152
R189, K190, D188, N187
R189, K190, D185, N208
Epi#27
D201, E253, D257, K258
D257, E253, D201, K267
Epi#28
I259, D257, Q254, E253, K267, F266, S260, R189
I259, D257, Q254, E253, K267, F266, S260, K258
Epi#29
L68, G108, L134, F170, E137
G135, N163, L134, F170, E137
Epi#30
G110, N107, A106, H71, L68, L104, L112
G108, N107, A106, H71, L68, P214, V213
G110, N107, A106, H71, P214, L68, L104
G110, N107, A106, H71, L68, L104, L134
Epi#33
Q12, Y220, V207, S222, S192, R189
190, F193, V207, S222, S192, R189
Q16, Y13, V207, S222, S192, R189
Epi#34
V26, W1, T27, G29, R28, S331, T332
W1, P21, T27, V26, R278, Y279, T255
Epi#35
G135, L134, S225, M221, I209
G110, L134, S225, M221, I209
G108, L134, S225, M221, I209
G162, L134, S225, M221, I209
Epi#37
A49, V52, L99, K54, N63
SAS: 309, Size 17.16: Y121, A127, L99, K54, N63
SAS: 307, Size 13.09: Y43, V52, L99, K54, N63
Epi#40
R189, G261, Y268, K258, S260
R189, G261, Y268, K258, T262
Epi#42
P3, S2, Q16, P21, R28, Q241
Epi#43
W199, Y196, G162, Q171, S140, L112, I115, T142
Epi#44
S145, D148, P176, W199, Y196, S167, G162, T169
S174, D201, P176, W199, Y196, S167, G197, T198
Epi#47
S330, S331, R28, V26, A22, Q16, N17, P21, S2
G242, S240, R28, V26, A22, Q16, N17, P21, S2
G29, S331, R28, V26, A22, Q16, N17, P21, S2
Epi#48
S2, D25, P21, P3, G86
S9, Q16, P21, P3, G86
Epi#50
R168, Y196, W199, T264, T198
D164, Y196, W199, T264, S260
Savinase:
Epi#01
L21, N18, P14, R19, K231, N232, S236, Q239, S234
L21, N18, P14, R19, K231, N232, S234, Q230, S24
L21, N18, P14, R19, K231, N232, S234, Q230, T22
Epi#02
T254, N255, A188, R164, S158, Y186, R180, V197
T249, N263, Q12, R10, P14, R19, R269
T249, N263, S9, R10, P14, R19, R269
Epi#03
K27, A86, I43, Y89
Epi#04
K229, S234, Q230, K231, R269, A266
K27, S24, Q230, K231, R269, A15
K231, S234, Q239, R241, N246, A248
Epi#05
G187, A188, N255, T254, G252, S250, T249, L251
G189, A188, N255, T254, G252, S250, T249, L261
Epi#06
G252, N179, D175, S182, S154, P127
A188, N255, D191, R164, S158, P127
A188, N255, D191, R164, S128, P127
Epi#08
A131, E134, S139, A106, F49
A166, E134, S139, A106, F49
Epi#09
S103, T132, A131, E134, A166, R164, N167, S142, R143
Epi#10
D175, N177, N179, S182, F183, G155, R180
D175, N212, N153, S182, F183, G155, R180
Epi#11
F49, L94, I105, Q107, V102, E134
F49, K92, I105, Q107, V102, E134
Epi#12
Y161, E134
Y165, E134
Epi#13
S76, L73, P39, T207, A209, P204, S206, G205, Y208
S85, L73, P39, T207, A209, P204, S206, G205, Y203
Epi#16
R164, P127, Y161, G152, S158, N255, D191, A166, N167
R164, P129, Y161, G152, S158, N255, D191, A166, N138
Epi#17
A156, S158, R164, S128
A188, S158, R164, S126
Epi#18
N177, N179, R180, S182, G155, S154, A156, S158
N177, N178, R180, S182, G155, S154, N153, F183
Epi#19
D175, N179, S182, Q185, R180, L256
D175, N179, S182, Q185, R180, L251
I240, W235, S234, Q239, R241, K245
D175, N179, S182, Q185, R180, G252
Epi#23
R143, N114, E110, S139, Q135, A131
R143, N115, E110, N138, Q135, A131
Epi#24
D58, G59, E53, P51, F49, P54, Q57
D58, G59, E53, P51, S48, P54, Q57
D58, G59, E53, P54, S55, F49, Q107
Epi#25
R19, R269, E265, N18
R269, R19, E265, N18
Epi#28
V102, Q107, F49, E53, K92, Q57, G46, R44
A47, Q107, F49, E53, K92, Q57, G46, R44
V50, Q107, F49, E53, K92, Q57, G46, R44
Epi#29
I77, N74, L41, R44, E87
V4, N74, L41, R44, E87
G20, N18, L21, R19, E265
Epi#30
G59, N60, S97, H62, L94, P51, P54
G98, N60, S97, H62, L94, P51, P54
Epi#31
L256, R180, N178, R10, W6, V197, D175
L251, R180, N178, R10, W6, V197, D175
Epi#33
Q107, F49, P51, S48, S55, K92
Q107, F49, P54, S55, A47, K92
Epi#34
V102, P129, S128, G125, R164, Y161, P127
V102, P129, S126, G125, R164, S158, P127
Epi#37
T254, A188, L256, R180, N177
T254, A188, L256, R180, N179
Epi#38
L94, G59, E53, A96, N60, P204, S206
L94, G59, E53, A96, N60, P204, S36
Epi#39
A131, E134, L133, T132, P129, G125, L124
A166, E134, L133, T132, P129, G125, L124
Epi#40
R44, L41, G78, T207, P39, T37
R19, L21, G20, T22, K231, S234
R180, L256, G252, T254, A188, S158
Epi#41
P127, Y161, L133, V102, S99
P127, Y161, L133, V102, S103
P127, Y161, L133, V102, S101
P127, Y161, L133, V102, S126
Epi#42
L73, P84, S85, N74, H17, P14, R19, R269
L80, P5, S3, N74, H17, P14, R19, R269
L21, P84, S85, N74, H17, P14, R19, R269
Epi#43
105, W111, A47, G46, Q57, S36, L41, I43, T37
Epi#44
S126, R164, P127, Y161, S158, A188, T254
S128, R164, P129, Y161, S158, A188, T254
Epi#46
A15, R269, R19, P14, N18, G20
A266, R269, R19, P14, N18, A15
Epi#48
S55, Q57, P54, P51, G52
E53, Q57, P54, P51, G52
Epi#50
R10, W6, S3, S76
R241, W235, S234, P233
R10, W6, V4, S9
Epi#51
Q239, H243, T247, R269, R19, K231, W235
R19, H17, E265, R269, K231, S234, W235
Epi#52
A15, S9, R10, W6, N198, Q176
A15, S9, R10, W6, N198, Q200
Amylase SP722:
Epi#02
T419, N423, P422, F396, T5, Y398, R393, R37
T419, N418, P422, F396, T5, Y398, R393, R37
Epi#03
L313, K311, H408, I410, Y404
L313, K311, H407, I410, Y404
Epi#04
R171, S170, Q172, R176, N175, I177
R181, Y135, S132, Q129, R127, N128, I131
Epi#05
G184, A186, N128, P124, G196, S193, H240, L201
G184, A186, N128, P124, G196, R127, S193, Y198
Epi#06
G147, N150, D154, T151, R148, P146
G149, N150, D154, T151, R148, P146
Epi#07
G149, T151, D154, S153, R219, V214, P211, D207
Epi#08
K311, D406, A310, H407, F405
K311, D308, A310, H408, F405
Epi#09
T461, R485, K484, N423, T419, N418
R485, K484, N423, T420, T419
Epi#10
E260, N255, R254, T251, F245, R218, R219
T419, N423, N395, T5, F396, R393, R37
E260, T257, N255, T251, F245, R218, R219
Epi#11
F173, I177, Q174, D166
L201, I177, Q174, D166
Epi#12
Y363, E360
Y398, E360
Y198, E194
Epi#13
H16, L17, P375, A381, P380, S378, P336, W15
H16, L17, P375, A381, P380, S378, P336, G337
H16, L17, P375, A381, P380, S378, P336, L340
Epi#15
N457, P459, D453, I454, K458, G456
K458, P459, D453, I454, T455, A384
N457, P459, D453, I454, K458, G460
Epi#16
Q319, P322, Y363, R359, Q401, D403, D406, A310, N314
Q319, P322, Y363, G362, R359, D403, D406, A310, N314
Q319, P322, Y363, R359, R415, D403, D406, A310, N314
Epi#17
A91, S32, R28, S87
A91, S87, R82, S83
Epi#18
R485, V450, G448, T463, T461, H452, V462
N126, N128, R127, G196, Y198, S193, N195, N125
N25, R26, R28, S87, I89, A91, H90, N94
Epi#19
D20, N54, S52, Q53, R76, G73
D20, N19, Q22, Q84, R76, G73
D29, N25, Q22, Q84, R28, A91
Epi#20
K385, P350, L355, L313, K311, D308, G305, D432
Epi#22
D183, A186, D209, W189, K242
D183, A186, D209, W189, E190
D183, A186, D209, P211, E212
D209, A186, D183, Y160, W159
D183, A186, D209, W187, W189
Epi#23
R415, N418, E416, N445, Q444, A466
K446, N445, E416, Y441, Q444, A466
Epi#24
D387, K385, A381, P375, S378, P380, K383
E341, G337, E338, P336, S378, A381, K385
D333, G337, E341, P336, S378, A381, K385
Epi#25
R485, H452, I454, E391, N36
R485, K484, I454, E391, N395
Epi#26
W167, S170, E138, W140, V117, G182, D183
W167, S170, E138, W140, V165, E121, D134
W167, S170, E138, W140, V165, E121, E130
Epi#27
E212, E216, D154, K156
E216, E212, D209, K242
Epi#28
L201, D166, Q172, W167, E138, W140, Q169, S170, R171
L201, D166, Q169, W140, E138, W167, F173, S170, R171
L201, D166, Q174, W167, E138, W140, Q169, S170, R171
Epi#29
V214, N215, L217, R219, E222
G96, H90, L228, R82, E86
V214, R219, L217, R218, E212
Epi#30
G456, N457, H452, K484, I454, P459
G362, M323, S287, H324, K320, P322, V318
G362, M323, S287, H321, K320, P322, V318
G460, N457, H452, K484, I454, P459
Epi#31
L217, R219, N215, R218, F245, V214, D248
L217, R219, N215, R218, F245, M208, D209
Epi#33
K383, Y372, P375, S378, A381, K385
K383, Y372, P380, S378, A381, K38
Epi#34
W439, W469, T463, V450, R485, T461, P459
W439, W469, T463, V462, R485, T461, P459
Epi#37
T251, R218, L217, R219, N215
P211, V214, L217, R219, N215
A256, R218, L217, R219, N215
Epi#38
G435, E437, N475, P434, S478
G436, E437, N475, P434, S478
Epi#39
E338, H16, T376, P336, G337, L340
E14, H16, T376, P336, G337, L340
Epi#40
A384, A381, Y372, P375, S378
A384, A381, Y372, P375, T376
Epi#41
P375, L17, V56, S52
Epi#42
S378, P380, Y372, A381, A384, P375
S378, P375, Y372, A381, A384, P388
S378, P375, Y372, A381, A384, T455
Epi#45
K72, P146, F69, Y64, R148, D154, G149
K311, H408, F405, N409, D432, G304
D406, H408, F405, N409, D432, G304
Epi#46
Y398, R393, R37, P388, Q394, G7
Y398, R359, R393, P388, G456, G460
Y398, R393, R37, P388, Q394, G38
Epi#47
A256, G258, E260, N255, R254, F262, N285, R247, S244
S193, Y198, E194, N125, R127, Q129, N123, R176, P124
Epi#48
S378, H377, P380, P375, V379
H16, H377, P375, P380, V379
Epi#49
D55, W15, L17, P18, Q22, Q84, T81, N25
D55, W15, L17, P18, Q22, Q84, T81, K78
Epi#50
D467, Y441, W439, W469, T463
D406, Y404, W439, W469, T463
D183, Y160, W159, W140, T114
D403, Y441, W439, W469, T463
Epi#51
D406, H408, D308, K311, L313, Q319, H321
Epi#52
W140, A113, E138, R171, W167, N175, Q174
W140, A113, E138, R171, W167, D166, Q172
Amylase AA560:
Epi#01
L390, P388, P350, K383, K385, N457, S478, R458, T461
L390, P388, P350, K383, K385, N457, S478, R458, T452
L390, P388, P350, K383, K385, N457, S478, R458, T455
Epi#02
L390, K395, Q394, R393, T5, Y398, R359, R400
L173, K172, S170, T136, Y135, R118, R181
L173, R171, S170, T136, Y135, R118, R181
L390, K395, Q394, R393, T5, Y398, R400, R415
Epi#03
K438, H407, I410, Y404
Epi#04
K172, S170, Q169, R171, N174, L173
R171, S170, Q169, K172, N175, I177
Epi#05
G456, A459, R458, T461, G460, T452, T463, V450
G456, A459, R458, T452, G460, T461, T463, G448
Epi#06
A51, N54, D20, R76, Q71, P146
G73, A51, D55, S52, K72, P146
Epi#07
G456, T455, S384, D387, R393, P388, D453
Epi#08
K259, S255, V222, H252, F245
K259, G258, A256, H252, F245
Epi#09
N128, V131, R176, D166, K172, N175, N174, R171
Epi#10
467, N445, R444, F441, R415, R400
D467, A466, R444, F441, R415, R400
Epi#11
F69, K72, I75, Q53, V56, D55
Epi#12
Y16, E337
Y363, E360
Y198, E194
Epi#15
K385, P388, D453, I454, R458, A459
K385, P388, D387, I454, T452, A459
K385, P388, D387, I454, R458, G456
Epi#17
A87, S29, R28, S32
A91, S29, R28, S32
Epi#18
N445, R444, A466, T463, T461, N471, N437
N445, R444, A466, T463, T461, T452, V450
Epi#19
166, W167, S170, Q169, R171, K172
E138, W167, S170, Q169, R171, K172
E134, T136, S170, Q169, R171, K172
Epi#22
D209, P211, D207, Y160, D183
Epi#23
R400, N418, E416, N445, Q449, A466
R82, N83, E68, N70, F69, P146
Epi#24
E134, G133, E130, P124, R176, L173, K172
E134, K179, E130, P124, R176, L173, K172
Epi#25
R444, K446, W469, D467, N445
R171, K172, W167, D166, N175
R171, K172, W167, D166, N174
Epi#26
W167, S170, E138, W140, V165, E121, E130
W167, S170, E138, W140, V165, E121, E134
W167, S170, E138, W140, V165, E121, D166
Epi#27
E130, E121, D166, K172
D36, E391, D387, K385
E134, E121, D166, K172
Epi#28
L201, D166, Q169, W140, E138, K172, S170, R171
L173, D166, Q169, K172, E138, W167, S170, R171
Epi#29
V131, R176, L173, R171, E138
I177, N175, L173, R171, E138
I177, N174, L173, R171, E138
Epi#30
I39, N33, S29, H23, P18, L17, P375
G38, N33, S29, H23, L17, P375, P380
G362, M323, S287, H321, Q319, P322, V318
G417, N423, A420, H421, K395, L390, P388
G21, N25, S29, H23, P18, L17, P375
G399, N418, A420, H421, K395, L390, P388
Epi#31
L173, R171, N174, R176, W167, M202, I177, D166
L173, R171, N174, R176, W167, V165, I177, D166
Epi#33
K108, Y58, V56, S52, A51, K72
Epi#34
W439, W469, T463, V450, G460, T452, T461
W15, P18, T376, G378, P375, Y372, S384
W469, W439, S473, G460, R458, T461, T463
Epi#37
P124, R176, L173, K172, N175
P124, R176, L173, R171, N174
Epi#40
R400, G399, Y396, P422, T419
R400, G417, Y396, P422, T419
Epi#41
P375, Y16, L17, V56, S52
P18, Y16, L17, V56, S52
Epi#42
P350, S478, G433, H408, R310, Q311
P322, S287, N285, H324, R320, Q319
P322, S287, G362, H321, R320, Q319
Epi#44
L17, D20, P18, W15, Y368, A381, G378, T376
L340, D333, P336, W15, Y368, A381, G378, P375
Epi#45
K72, P146, F69, Y64, N150, D144, G147
D112, P146, F69, Y64, N150, D144, G149
Epi#46
Y398, R359, R393, P388, G456, A459
Y363, R359, R393, P388, Q394, G7
Y363, R359, R393, P388, Q394, G38
Epi#47
I75, E68, R76, N83, R82, Q84, N90, R28, S29
G133, E134, E130, V131, R176, L173, N174, R171, S170
Epi#48
S384, K383, P380, P375, G378
E337, H377, P380, P375, V379
Epi#50
R444, W469, W439, S473, T461
D183, Y160, W159, W140, T114
Epi#51
R320, H321, Q319, P322, H324, H286
Epi#52
W140, A113, E138, R171, W167, D166, Q169
W140, A113, E115, R118, W159, T114, Q169
Protease A:
Epi#01
L21, N18, P14, R19, K237, N238, S242, Q245, S240
L21, N18, P14, R19, K237, N238, S240, Q236, S24
Epi#02
T255, N269, Q12, R10, P14, R19, R275
T255, N269, S9, R10, P14, R19, R275
Epi#03
K27, A88, I44, Y91
Epi#04
K235, S240, Q236, K237, R275, A15
K27, S24, Q236, K237, R275, A15
K237, S240, Q245, R247, N252, A254
R145, S141, Q137, Y171, N173, A172
Epi#06
G61, N62, D60, T38, Q59, P55
G211, P210, D60, T38, Q59, P55
A98, N62, D60, T38, Q59, P55
G100, N62, D60, T38, Q59, P55
Epi#08
A131, E136, S141, A108, F50
A172, E136, S141, A108, F50
A98, E54, G53, V51, F50
Epi#09
S162, S170, A172, N173, V244, H249, N252, S256, T260
S259, S256, T260, N261, L262, R186, N185, S188, N155
S162, S170, A172, N173, V244, H249, N248, N252, T255
S156, S162, N261, S259, L262, R186, N185, S188, N155
Epi#10
D181, N183, N185, S188, F189, G157, R186
D181, N218, N155, S156, F189, G157, R186
Epi#12
Y171, E136
Y91, E89
Epi#13
S78, L75, P40, T213, A215, P210, S212, G211, Y209
S87, L75, P40, T213, A215, P210, S212, G211, Y214
Epi#16
L262, P194, Y192, G195, S162, N261, D197, A172, N140
L262, P194, Y192, G157, S162, N261, D197, A172, N173
L262, P194, Y192, G161, S162, S170, D197, A172, N173
Epi#17
A138, S141, R145, S144
A108, S141, R145, S144
Epi#18
N185, N183, R186, L262, S259, T260, P194, N261
N185, N183, R186, L262, Y192, T260, P194, S162
Epi#19
I246, W241, S240, Q245, R247, K251
D181, N185, S188, Q191, R186, L262
Epi#23
R145, N116, E112, S141, Q137, A138
R145, N117, E112, S141, Q137, A108
Epi#24
E136, G133, A131, P129, S103, F50, Q109
E136, G132, A131, P129, S103, A108, Q137
D60, G61, E54, P52, F50, P55, Q59
Epi#25
R275, R19, E271, N18
Epi#28
G20, H17, Q12, E271, L21, Q236, S240, K237
A15, H17, Q12, E271, L21, Q236, S240, K237
Epi#29
V244, Q245, L148, R145, E112
V244, N173, L148, R145, E112
Epi#30
G61, N62, A98, H64, L96, P52, P55
G20, N18, A15, H17, S87, L75, P40
I79, N76, S87, H17, Q12, P14, V4
G100, N62, A98, H64, L96, P52, P55
Epi#31
L262, R186, N184, R10, W6, V203, D181
L257, R186, N184, R10, W6, V203, D181
Epi#33
Q109, F50, P52, S49, S56, K94
Q109, F50, P55, S56, A48, K94
Epi#34
W241, P239, S242, G146, R145, S141, S144
I165, P194, T260, G258, R186, S188, S156
V104, P129, S130, G127, G102, S101, S99
V244, W241, S242, G146, R145, S141, S144
I165, P194, S170, G127, P129, S130, S103
Epi#37
P14, A15, L21, R19, N18
T143, R145, L148, R247, N252
T143, V244, L148, R145, N116
Epi#38
L96, G97, E54, A98, N62, P210, S212
L96, G97, E54, A98, N62, P210, S37
Epi#39
A15, E271, H17, R19, P14, G20, L21
A254, E271, H17, R19, P14, G20, L21
A272, E271, H17, R19, P14, G20, L21
Epi#40
R186, L257, G258, T260, P194, S162
R186, L262, G161, Y192, P194, T260
Epi#41
P194, Y192, L262, S259
P194, Y192, L196, S162
Epi#42
L82, P5, S3, N76, H17, P14, R19, R275
L82, P5, S9, Q12, H17, P14, R19, R275
Epi#43
W113, A48, G47, Q59, S37, L42, I44, T38
Epi#44
V244, R247, D197, P194, Y192, S162, G195, T260
V244, R247, D197, P194, Y192, S170, G195, T260
S56, Q59, D60, P210, Y214, S212, G211, T38
S56, Q59, D60, P210, Y209, S212, G211, T38
Epi#46
A15, R275, R19, P14, N18, G20
A272, R275, R19, P14, N18, G20
A272, R275, R19, P14, N18, A15
Epi#47
S130, A131, E136, N173, A172, N140, R145, S144
S105, A131, E136, N173, A172, N140, R145, S144
Epi#48
E54, Q59, P55, P52, G53
S56, Q59, P55, P52, G53
S49, Q59, P55, P52, G53
Epi#50
R10, W6, S3, S78
R10, W6, V4, S9
R10, W6, V203, S188
Epi#51
Q245, H249, T253, R275, K237, S240, W241
R19, H17, E271, R275, K237, S240, W241
R145, H120, K27, S24, K237, S240, W241
R145, H120, K235, K237, P239, S240, W241
Epi#52
A15, S9, R10, W6, N204, Q206
A15, S9, R10, W6, N204, Q182
Alcalase:
Epi#01
L10, P5, P9, K15, K12, N269, S251, R249, T253
L82, P5, P9, K15, K12, N269, S251, R249, T253
Epi#02
T115, N141, A144, R145, S242, R247, R249
A138, N141, A144, R145, S242, R247, R249
Epi#03
L196, K170, A129, I165, Y167
L196, K170, A194, I165, Y171
Epi#04
R145, Y143, S173, Q137, K136, T133, A134
K170, Y167, S132, Q137, K136, N141, A144
Epi#05
G53, A52, F50, G102, S105, S103, Y104
G53, A52, F50, G102, S101, S103, Y104
Epi#06
A24, N25, Q120, R145, S242, P239
A144, N141, D140, R145, S242, P239
Epi#08
K265, E197, S260, A194, F261
A56, E54, G53, A52, F50
Epi#10
T162, N161, N163, A194, F261, G264, K265
E195, N161, N163, S158, F261, G258, K265
Epi#12
Y57, E54
Y262, E197
Epi#13
S38, A37, P40, T213, A215, H64, L217, G204, Y206
S38, A37, P40, T213, A215, H64, S98, G100, G61
S87, L75, P40, T213, A215, H64, L217, G204, Y6
Epi#16
L10, P9, Y6, G204, S182, N183, D181, A187, N185
Q2, P5, Y206, G204, S182, N183, Q181, A203, N218
L10, P9,Y6, G204, S182, N183, D181, A187, N155
Epi#17
A144, S244, R247, S252
A272, S252, R249, S244
A144, S244, R249, S251
A254, S252, R249, S244
Epi#18
N141, R145, A144, Y143, S244, N248, S252
Epi#19
N248, S244, Q245, R249, A272
N240, S242, Q245, R249, A254
N240, S242, Q245, R249, L241
Epi#22
D76, L82, D14, A18, K15
D181, L10, D14, A18, K15
Epi#23
K27, N117, E112, N141, Q137, A134
K27, N117, E112, N141, Q137, A138
K27, N117, E112, S109, F50, A52
Epi#24
D120, K27, A24, P86, F21, A18, K15
D14, K22, A24, P86, F21, A18, K15
D76, K22, A24, P86, S87, F21, K15
Epi#25
R249, R247, E197, E195
Epi#27
D172, E195, E197, K265
E197, E195, D172, K136
D172, E197, E195, K170
Epi#28
A18, D14, Q19, K15, E271, K12, Q17, S87, D76
V4, D14, Q17, K12, E271, K15, F21, A18, K22
Epi#29
L257, K265, L196, F261, E195
G53, N97, L96, F50, E54
Epi#30
G146, L241, S242, H238, K237, P239, L235
G146, L241, S236, H238, S242, P239, L235
Epi#33
K15, F21, P86, S87, A24, K27
K27, Y91, V45, S89, A24, K22
Epi#34
V4, P5, T3, G80, P40, S38, T211
V108, W113, T116, G118, R145, Y143, S244
V26, P239, S242, G146, R145, T115, T116
Epi#36
A52, A56, A48, V51, G102, Y104, S105, V108, A138, A134
A52, A56, A48, V51, G102, Y104, S103, V108, A134, A138
Epi#37
Y262, A194, L196, K265, Y256
Y263, R186, L257, K265, Y256
Y256, A254, L257, K265, Y262
Epi#40
R186, L257, A254, Y256, K265, S252
R186, L257, G258, Y256, K265, S260
Epi#41
Y256, L257, S260
Y256, L257, S259
Epi#42
L235, P239, S242, N248, R249, Q275
L241, P239, S242, Q245, R249, Q275
Epi#44
S132, Q137, D140, Y143, A144, A138, T133
V108, Q137, D140, Y143, A144, A138, T133
S173, Q137, D140, Y143, A144, A138, T133
Epi#48
Q19, K15, P9, P5, V4
E271, K15, P9, P5, V4
Protease B:
Epi#05
SAS: 454, Size 24.86: G189, A188, R164, P127, G125, S99
SAS: 452, Size 15.92: G189, A188, R164, P127, G125, S128
SAS: 451, Size 24.86: G157, A188, R164, P127, G125, S99
SAS: 449, Size 15.92: G157, A188, R164, P127, G125, S128
SAS: 445, Size 23.31: G189, A166, R164, P127, G125, S99
Epi#09
SAS: 446, Size 15.76: T254, G189, A166, R164, A188, S158
SAS: 312, Size 15.90: T22, G20, L21, R19, A15, S9
Epi#10
SAS: 460, Size 17.32: D175, N177, N179, S182, F183, G155, R180
SAS: 437, Size 16.70: D211, N212, N153, S182, F183, G155, R180
SAS: 424, Size 13.75: D175, N212, N153, S182, F183, G155, R180
SAS: 417, Size 16.70: D211, N212, N153, S154, F183, G155, R180
SAS: 404, Size 15.83: D175, N212, N153, S154, F183, G155, R180
Epi#12
SAS: 309, Size 13.46: P127, Y161, E134, P129
SAS: 292, Size 9.37: R164, Y161, E134, P129
SAS: 287, Size 18.66: P127, Y161, E134, N138
SAS: 284, Size 16.85: P127, Y161, E134, N167
SAS: 275, Size 11.53: S128, Y161, E134, P129
Epi#17
SAS: 275, Size 15.84: A188, S158, R164, S126
SAS: 225, Size 12.79: A156, S158, R164, S126
Epi#18
SAS: 444, Size 16.32: S250, K245, S259, L256, A188, T254, L251
SAS: 397, Size 14.14: S250, K245, S259, L256, G252, T254, L251
SAS: 397, Size 14.14: S250, K245, S259, L251, G252, T254, L256
SAS: 397, Size 14.14: S259, K245, S250, L251, G252, T254, L256
SAS: 396, Size 21.52: S158, R164, S126, V102, G100, S99, L124
Epi#19
SAS: 295, Size 15.06: D175, W6, S9, Q12, R10
SAS: 278, Size 21.23: E110, T141, S236, Q239, R241
Epi#23
SAS: 486, Size 19.88: R143, N114, E110, S139, Q135, A131
SAS: 473, Size 18.68: R19, N18, E265, L21, Q230, P233
SAS: 468, Size 15.74: R164, N167, E134, S139, Q135, A131
SAS: 463, Size 13.77: R164, N167, E134, S130, Q135, A131
SAS: 461, Size 21.98: R44, N42, E87, S24, Q230, P233
Epi#28
SAS: 520, Size 19.27: V102, Q107, W111, E110, Q135, S139, R143
SAS: 492, Size 24.70: V102, Q107, F49, E53, Q57, G46, R44
SAS: 480, Size 22.76: V50, Q107, W111, E110, Q135, S139, R143
SAS: 452, Size 19.08: V50, Q107, F49, E53, Q57, G46, R44
SAS: 441, Size 24.70: V102, Q107, E110, W111, F49, G46, R44
Epi#29
SAS: 239, Size 11.49: G20, N18, L21, E265
SAS: 224, Size 11.49: G20, R19, L21, E265
SAS: 179, Size 16.62: I4, P14, L21, E265
SAS: 175, Size 11.49: G20, K231, L21, E265
SAS: 153, Size 18.96: G25, Q230, L21, E265
Epi#30
SAS: 308, Size 24.27: G20, L21, A15, H17, S85, L73, P39
Epi#31
SAS: 363, Size 21.72: L256, R180, N178, R10, W6, V197, D211
SAS: 352, Size 22.95: L251, R180, N178, R10, W6, V197, D211
SAS: 350, Size 21.62: L256, R180, N178, R10, W6, V197, D175
SAS: 339, Size 17.75: L251, R180, N178, R10, W6, V197, D175
Epi#34
SAS: 430, Size 18.33: V238, W235, S236, G144, R143, S139, S142
SAS: 430, Size 18.33: V238, W235, S236, G144, R143, S142, S139
SAS: 420, Size 13.98: V238, W235, S236, G144, R143, S142, T141
SAS: 420, Size 13.98: V238, W235, S236, G144, R143, T141, S142
SAS: 352, Size 18.33: V238, W235, S236, G144, R143, S139, T141
Epi#37
SAS: 415, Size 23.06: T254, A188, L256, R180, N177
SAS: 374, Size 18.08: T254, A188, L256, R180, N179
SAS: 335, Size 19.96: T254, A188, L256, R180, N178
Epi#39
SAS: 425, Size 16.00: A166, E134, R164, P127, G125, L124
SAS: 421, Size 16.36: A131, E134, R164, P127, G125, L124
SAS: 400, Size 16.00: A166, E134, R164, P129, G125, L124
SAS: 396, Size 16.36: A131, E134, R164, P129, G125, L124
SAS: 359, Size 16.00: A166, E134, T132, P129, G125, L124
Epi#40
SAS: 358, Size 15.76: A166, G189, Y186, A188, T254
SAS: 352, Size 15.76: A166, G189, T254, A188, S158
SAS: 326, Size 11.62: A96, G59, T56, P54, S55
SAS: 322, Size 15.30: G98, G59, T56, P54, S55
SAS: 318, Size 17.81: A188, G189, Y186, A156, S182
Epi#42
SAS: 528, Size 16.22: L21, P14, S9, Q12, H17, R19, R269
Epi#44
SAS: 401, Size 15.10: L256, R180, Y186, S158, A188, T254
SAS: 393, Size 15.52: L256, R180, Y186, A188, G189, T254
SAS: 390, Size 18.46: L251, R180, Y186, S158, A188, T254
SAS: 382, Size 16.23: L251, R180, Y186, A188, G189, T254
SAS: 376, Size 22.23: V197, R180, Y186, S158, A188, T254
Epi#46
SAS: 559, Size 12.63: A15, R269, R19, P14, N18, G20
Epi#53
SAS: 298, Size 9.48: W235, S234, Q230, K231
SAS: 298, Size 18.05: W235, S234, Q239, K245
SAS: 289, Size 9.48: W235, P233, Q230, K231
SAS: 283, Size 9.61: W235, S234, Q239, K229
SAS: 255, Size 14.51: W235, S236, Q239, K245
ProteaseC:
Epi#05
SAS: 445, Size 23.34: G189, A166, R164, P127, G125, S99
SAS: 445, Size 24.90: G189, A188, R164, P127, G125, S99
SAS: 433, Size 24.90: G157, A188, R164, P127, G125, S99
SAS: 427, Size 15.89: G189, A188, R164, P127, G125, S128
SAS: 427, Size 15.50: G189, A166, R164, P127, G125, S128
Epi#09
SAS: 463, Size 15.74: T254, G189, A166, R164, A188, S158
SAS: 425, Size 15.74: D191, G189, A166, R164, A188, T254
SAS: 384, Size 13.57: D191, G189, A166, R164, A188, S158
Epi#10
SAS: 445, Size 17.28: D175, N177, N179, S182, F183, G155, R180
SAS: 431, Size 13.75: D175, N212, N153, S182, F183, G155, R180
SAS: 403, Size 15.83: D175, N212, N153, S154, F183, G155, R180
SAS: 387, Size 16.14: D175, N178, N179, S182, F183, G155, R180
SAS: 373, Size 16.76: D175, N212, N153, A156, F183, G155, R180
Epi#12
SAS: 292, Size 13.45: P127, Y161, E134, P129
SAS: 287, Size 9.30: R44, Y89, E87, N42
SAS: 284, Size 9.35: R164, Y161, E134, P129
SAS: 282, Size 9.35: R164, Y165, E134, P129
SAS: 272, Size 16.85: P127, Y161, E134, N167
Epi#16
SAS: 547, Size 20.59: R164, P129, Y165, G189, S158, N255, D191,
A166, N167
SAS: 543, Size 23.80: R164, P129, Y165, G189, S158, N255, D191,
A166, N138
Epi#17
SAS: 267, Size 15.84: A188, S158, R164, S126
SAS: 231, Size 12.82: A156, S158, R164, S126
Epi#18
SAS: 449, Size 16.85: S182, R180, L256, A188, T254, L251
SAS: 426, Size 21.97: S126, R164, S158, A188, T254, L256
SAS: 407, Size 15.92: S182, R180, L251, G252, T254, L256
SAS: 407, Size 15.92: S182, R180, L256, G252, T254, L251
SAS: 391, Size 18.26: S182, R180, L256, G252, S250, L251
Epi#19
SAS: 293, Size 15.04: D175, W6, S9, Q12, R10
SAS: 291, Size 17.13: D191, N242, S236, Q239, R241
SAS: 273, Size 21.24: E110, T141, S236, Q239, R241
Epi#23
SAS: 463, Size 19.84: R143, N114, E110, S139, Q135, A131
SAS: 451, Size 15.68: R164, N167, E134, S139, Q135, A131
SAS: 443, Size 21.95: R44, N42, E87, S24, Q230, P233
SAS: 440, Size 22.70: R143, N115, E110, S139, Q135, A131
SAS: 431, Size 15.11: R44, N42, E87, S85, L73, P39
Epi#28
SAS: 402, Size 18.79: G59, Q57, E53, F49, G46, R44
SAS: 384, Size 20.81: A96, Q57, E53, F49, G46, R44
SAS: 376, Size 18.79: A47, Q57, E53, F49, G46, R44
Epi#31
SAS: 348, Size 21.63: L256, R180, N178, R10, W6, V197, D175
SAS: 342, Size 17.75: L251, R180, N178, R10, W6, V197, D175
Epi#33
SAS: 399, Size 18.88: Q107, Y102, P129, S126, R164
SAS: 355, Size 15.95: Q135, Y165, P129, S126, R164
Epi#34
SAS: 424, Size 18.37: V238, W235, S236, G144, R143, S139, S142
SAS: 424, Size 18.37: V238, W235, S236, G144, R143, S142, S139
SAS: 408, Size 14.02: V238, W235, S236, G144, R143, S142, T141
SAS: 408, Size 14.02: V238, W235, S236, G144, R143, T141, S142
SAS: 346, Size 18.37: V238, W235, S236, G144, R143, T141, S139
Epi#37
SAS: 405, Size 23.05: T254, A188, L256, R180, N177
SAS: 364, Size 18.08: T254, A188, L256, R180, N179
SAS: 347, Size 19.96: T254, A188, L256, R180, N178
Epi#40
SAS: 368, Size 15.74: A166, G189, T254, A188, S158
SAS: 362, Size 15.74: A166, G189, Y186, A188, T254
SAS: 326, Size 17.80: A188, G189, Y186, A156, S182
SAS: 326, Size 23.72: A166, G189, Y186, A156, S182
SAS: 326, Size 17.80: G189, A188, Y186, A156, S182
Epi#41
SAS: 232, Size 19.49: P204, Y208, L211, V197, S210
Epi#44
SAS: 445, Size 22.71: V238, R241, D191, Y186, S158, A188, T254
SAS: 429, Size 21.14: V238, R241, D191, Y186, A188, G189, T254
SAS: 410, Size 22.71: V238, R241, D191, Y186, S158, G189, T254
SAS: 404, Size 23.33: V238, R241, D191, Y257, S250, G252, T254
SAS: 382, Size 23.33: V238, R241, D191, Y257, S253, G252, T254
Epi#46
SAS: 567, Size 12.67: A15, R269, R19, P14, N18, G20
Epi#53
SAS: 305, Size 9.43: W235, S234, Q230, K231
SAS: 303, Size 9.53: W235, S234, Q239, K229
SAS: 276, Size 9.43: W235, P233, Q230, K231
SAS: 259, Size 9.43: W235, S234, Q230, K229
SAS: 233, Size 9.53: W235, S236, Q239, K229
ProteaseD:
Epi#05
SAS: 453, Size 24.94: G189, A188, R164, P127, G125, S99
SAS: 449, Size 23.37: G189, A166, R164, P127, G125, S99
SAS: 442, Size 24.94: G157, A188, R164, P127, G125, S99
SAS: 439, Size 15.91: G189, A188, R164, P127, G125, S128
SAS: 435, Size 15.50: G189, A166, R164, P127, G125, S128
Epi#09
SAS: 448, Size 15.77: T254, G189, A166, R164, A188, S158
Epi#10
SAS: 460, Size 17.32: D175, N177, N179, S182, F183, G155, R180
SAS: 428, Size 13.76: D175, N212, N153, S182, F183, G155, R180
SAS: 403, Size 15.83: D175, N212, N153, S154, F183, G155, R180
SAS: 391, Size 16.15: D175, N178, N179, S182, F183, G155, R180
SAS: 372, Size 16.77: D175, N212, N153, A156, F183, G155, R180
Epi#12
SAS: 302, Size 13.47: P127, Y161, E134, P129
SAS: 290, Size 9.39: R164, Y161, E134, P129
SAS: 282, Size 18.68: P127, Y161, E134, N138
SAS: 280, Size 16.87: P127, Y161, E134, N167
SAS: 270, Size 13.10: R164, Y161, E134, N138
Epi#17
SAS: 286, Size 15.87: A188, S158, R164, S126
SAS: 250, Size 12.76: A156, S158, R164, S126
Epi#18
SAS: 446, Size 16.31: S250, K245, S259, L256, A188, T254, L251
SAS: 406, Size 14.13: S250, K245, S259, L256, G252, T254, L251
SAS: 406, Size 14.13: S250, K245, S259, L251, G252, T254, L256
SAS: 406, Size 14.13: S259, K245, S250, L251, G252, T254, L256
SAS: 388, Size 14.13: S250, K245, S259, L256, G252, T249, L251
Epi#19
SAS: 319, Size 15.07: D175, W6, S9, Q12, R10
SAS: 276, Size 21.28: E110, T141, S236, Q239, R241
Epi#23
SAS: 497, Size 19.86: R143, N114, E110, S139, Q135, A131
SAS: 487, Size 15.77: R164, N167, E134, S139, Q135, A131
SAS: 478, Size 13.78: R164, N167, E134, S130, Q135, A131
SAS: 477, Size 18.16: R143, N138, E134, S139, Q135, A131
SAS: 472, Size 22.70: R143, N115, E110, S139, Q135, A131
Epi#28
SAS: 554, Size 22.17: A101, Q107, I102, E134, Q135, S139, R143
SAS: 532, Size 19.36: I102, Q107, W111, E110, Q135, S139, R143
SAS: 527, Size 22.79: V50, Q107, I102, E134, Q135, S139, R143
SAS: 509, Size 24.76: I102, Q107, F49, E53, Q57, G46, R44
SAS: 508, Size 22.17: A101, Q107, W111, E110, Q135, S139, R143
Epi#31
SAS: 355, Size 21.56: L256, R180, N178, R10, W6, V197, D175
SAS: 352, Size 17.71: L251, R180, N178, R10, W6, V197, D175
Epi#34
SAS: 457, Size 18.37: V238, W235, S236, G144, R143, S139, S142
SAS: 457, Size 18.37: V238, W235, S236, G144, R143, S142, S139
SAS: 447, Size 14.02: V238, W235, S236, G144, R143, S142, T141
SAS: 447, Size 14.02: V238, W235, S236, G144, R143, T141, S142
SAS: 374, Size 18.37: V238, W235, S236, G144, R143, T141, S139
Epi#37
SAS: 397, Size 23.08: T254, A188, L256, R180, N177
SAS: 361, Size 18.08: T254, A188, L256, R180, N179
SAS: 328, Size 19.98: T254, A188, L256, R180, N178
Epi#39
SAS: 425, Size 16.36: A131, E134, R164, P127, G125, L124
SAS: 423, Size 16.02: A166, E134, R164, P127, G125, L124
SAS: 399, Size 16.36: A131, E134, R164, P129, G125, L124
SAS: 397, Size 16.02: A166, E134, R164, P129, G125, L124
SAS: 379, Size 16.36: A131, E134, T132, P129, G125, L124
Epi#40
SAS: 354, Size 15.77: A166, G189, T254, A188, S158
SAS: 351, Size 15.77: A166, G189, Y186, A188, T254
SAS: 334, Size 17.81: G189, A188, Y186, A156, S182
SAS: 334, Size 17.81: A188, G189, Y186, A156, S182
SAS: 330, Size 14.42: A166, G189, Y186, A188, S158
Epi#41
SAS: 217, Size 19.46: P204, Y208, L211, V197, S210
Epi#44
SAS: 407, Size 15.10: L256, R180, Y186, S158, A188, T254
SAS: 404, Size 18.45: L251, R180, Y186, S158, A188, T254
SAS: 387, Size 15.52: L256, R180, Y186, A188, G189, T254
SAS: 384, Size 16.23: L251, R180, Y186, A188, G189, T254
SAS: 373, Size 22.26: V197, R180, Y186, S158, A188, T254
Epi#46
SAS: 545, Size 12.69: A15, R269, R19, P14, N18, G20
Epi#53
SAS: 306, Size 18.06: W235, S234, Q239, K245
SAS: 277, Size 9.52: W235, S234, Q239, K229
SAS: 276, Size 9.46: W235, S234, Q230, K231
SAS: 268, Size 9.46: W235, P233, Q230, K231
SAS: 258, Size 14.50: W235, S236, Q239, K245
ProteaseE:
Epi#05
SAS: 461, Size 15.49: G189, A166, R164, P127, G125, S128
SAS: 459, Size 15.90: G189, A188, R164, P127, G125, S128
SAS: 435, Size 15.49: G189, A166, R164, P127, G125, S126
SAS: 433, Size 15.49: G189, A166, R164, P129, G125, S128
SAS: 433, Size 15.86: G189, A188, R164, P127, G125, S126
Epi#06
SAS: 518, Size 14.10: G189, A188, D157, S158, R164, P127
SAS: 490, Size 15.98: G189, A188, D157, S158, R164, P129
SAS: 460, Size 14.60: G155, A156, D157, S158, R164, P127
SAS: 432, Size 17.71: G155, A156, D157, S158, R164, P129
Epi#09
SAS: 482, Size 15.78: T254, G189, A166, R164, A188, S158
SAS: 311, Size 15.91: T22, G20, L21, R19, A15, S9
Epi#10
SAS: 455, Size 17.26: D175, N177, N179, S182, F183, G155, R180
SAS: 406, Size 13.76: D175, N212, N153, S182, F183, G155, R180
SAS: 383, Size 16.16: D175, N178, N179, S182, F183, G155, R180
SAS: 381, Size 15.82: D175, N212, N153, S154, F183, G155, R180
SAS: 347, Size 16.78: D175, N212, N153, A156, F183, G155, R180
Epi#12
SAS: 310, Size 13.48: P127, Y161, E134, P129
SAS: 306, Size 9.40: R164, Y161, E134, P129
SAS: 297, Size 9.40: R164, Y165, E134, P129
SAS: 285, Size 16.90: P127, Y161, E134, N167
SAS: 281, Size 18.68: P127, Y161, E134, N138
Epi#16
SAS: 673, Size 19.67: R164, P127, Y161, G125, S126, S154, D157,
A188, N255
SAS: 664, Size 20.60: R164, P129, Y165, G189, S158, S154, D157,
A188, N255
SAS: 645, Size 20.60: R164, P129, Y161, G125, S126, S154, D157,
A188, N255
SAS: 636, Size 14.89: R164, P127, Y161, G125, S126, S154, D157,
A156, N153
SAS: 627, Size 17.25: R164, P129, Y165, G189, S158, S154, D157,
A156, N153
Epi#17
SAS: 305, Size 15.86: A188, S158, R164, S126
SAS: 270, Size 12.73: A156, S158, R164, S126
Epi#18
SAS: 590, Size 17.32: S250, K246, S259, L256, A188, T254, L251
SAS: 551, Size 16.26: S259, K246, S250, L251, G252, T254, L256
SAS: 551, Size 16.26: S250, K246, S259, L251, G252, T254, L256
SAS: 551, Size 16.26: S250, K246, S259, L256, G252, T254, L251
SAS: 518, Size 16.26: S250, K246, S259, L251, G252, S253, L256
Epi#23
SAS: 471, Size 19.86: R143, N114, E110, S139, Q135, A131
SAS: 467, Size 13.75: R164, N167, E134, S130, Q135, A131
SAS: 467, Size 15.76: R164, N167, E134, S139, Q135, A131
SAS: 451, Size 22.69: R143, N115, E110, S139, Q135, A131
SAS: 446, Size 19.99: R143, N138, E134, S130, Q135, A131
Epi#28
SAS: 505, Size 19.43: I102, Q107, W111, E110, Q135, S139, R143
SAS: 500, Size 22.22: A101, Q107, W111, E110, Q135, S139, R143
SAS: 499, Size 24.79: I102, Q107, F49, E53, Q57, G46, R44
SAS: 494, Size 24.56: A101, Q107, F49, E53, Q57, G46, R44
SAS: 441, Size 24.79: I102, Q107, E110, W111, F49, G46, R44
Epi#29
SAS: 216, Size 9.94: I43, R44, L41, E87
SAS: 209, Size 10.85: L73, N42, L41, E87
SAS: 200, Size 13.98: G46, R44, L41, E87
SAS: 199, Size 11.98: G45, R44, L41, E87
SAS: 197, Size 19.08: I77, N74, L41, E87
Epi#30
SAS: 318, Size 24.25: G20, L21, A15, H17, S85, L73, P39
SAS: 277, Size 24.25: G20, L21, A15, H17, S85, L41, P39
SAS: 258, Size 21.05: G20, L21, A15, H17, S85, L73, L41
Epi#31
SAS: 377, Size 21.62: L256, R180, N178, R10, W6, V197, D175
SAS: 370, Size 17.72: L251, R180, N178, R10, W6, V197, D175
Epi#33
SAS: 388, Size 15.92: Q135, Y165, P129, S126, R164
Epi#34
SAS: 420, Size 18.35: V238, W235, S236, G144, R143, S139, S142
SAS: 411, Size 13.98: V238, W235, S236, G144, R143, S142, T141
SAS: 341, Size 18.35: V238, W235, S236, G144, R143, S139, T141
Epi#37
SAS: 412, Size 23.05: T254, A188, L256, R180, N177
SAS: 378, Size 18.07: T254, A188, L256, R180, N179
SAS: 340, Size 20.00: T254, A188, L256, R180, N178
Epi#39
SAS: 445, Size 16.04: A166, E134, R164, P127, G125, L124
SAS: 432, Size 16.40: A131, E134, R164, P127, G125, L124
SAS: 417, Size 16.04: A166, E134, R164, P129, G125, L124
SAS: 404, Size 16.40: A131, E134, R164, P129, G125, L124
SAS: 376, Size 16.04: A166, E134, T132, P129, G125, L124
Epi#40
SAS: 374, Size 15.78: A166, G189, T254, A188, S158
SAS: 334, Size 15.78: A166, G189, Y186, A188, T254
SAS: 317, Size 11.62: A96, G59, T56, P54, S55
SAS: 312, Size 15.30: G98, G59, T56, P54, S55
SAS: 307, Size 15.49: G189, A166, Y165, P129, S128
Epi#41
SAS: 234, Size 19.50: P204, Y208, L211, V197, S210
SAS: 189, Size 19.50: P204, Y208, L211, V197, S215
Epi#42
SAS: 549, Size 16.42: L21, P14, S9, Q12, H17, R19, R269
Epi#44
SAS: 398, Size 15.10: L256, R180, Y186, S158, A188, T254
SAS: 391, Size 18.47: L251, R180, Y186, S158, A188, T254
SAS: 372, Size 15.51: L256, R180, Y186, A188, G189, T254
SAS: 371, Size 12.26: L256, R180, Y257, S250, G252, T254
SAS: 367, Size 15.51: L256, R180, Y186, S158, G189, T254
Epi#46
SAS: 575, Size 12.75: A15, R269, R19, P14, N18, G20
Epi#47
SAS: 491, Size 19.28: G45, E87, I43, R44, L41, N42, P39, S206
Epi#53
SAS: 202, Size 9.12: W235, P233, K231
SAS: 199, Size 9.12: W235, S234, K231
SAS: 182, Size 6.73: W235, P233, K229
SAS: 179, Size 7.76: W235, S234, K229
SAS: 131, Size 8.39: W235, S236, K229
Properase:
Epi#05
SAS: 456, Size 15.94: G189, A188, R164, P127, G125, S128
SAS: 453, Size 15.52: G189, A166, R164, P127, G125, S128
SAS: 451, Size 15.94: G157, A188, R164, P127, G125, S128
SAS: 427, Size 15.94: G189, A188, R164, P129, G125, S128
SAS: 424, Size 15.52: G189, A166, R164, P129, G125, S128
Epi#09
SAS: 480, Size 15.73: T254, G189, A166, R164, A188, S158
SAS: 302, Size 15.88: T22, G20, L21, R19, A15, S9
Epi#10
SAS: 470, Size 17.27: D175, N177, N179, S182, F183, G155, R180
SAS: 446, Size 13.75: D175, N212, N153, S182, F183, G155, R180
SAS: 420, Size 15.84: D175, N212, N153, S154, F183, G155, R180
SAS: 396, Size 16.09: D175, N178, N179, S182, F183, G155, R180
SAS: 380, Size 16.78: D175, N212, N153, A156, F183, G155, R180
Epi#12
SAS: 296, Size 9.36: R164, Y161, E134, P129
SAS: 295, Size 13.45: P127, Y161, E134, P129
SAS: 291, Size 9.36: R164, Y165, E134, P129
SAS: 271, Size 14.70: R164, Y161, E134, N102
SAS: 270, Size 13.45: P127, Y161, E134, N102
Epi#17
SAS: 283, Size 15.87: A188, S158, R164, S126
SAS: 241, Size 12.73: A156, S158, R164, S126
Epi#18
SAS: 474, Size 16.26: S250, K245, S259, L256, A188, T254, L251
SAS: 435, Size 14.14: S250, K245, S259, L256, G252, T254, L251
SAS: 398, Size 14.14: S259, K245, S250, L251, G252, S253, L256
Epi#19
SAS: 260, Size 21.26: E110, T141, S236, Q239, R241
Epi#23
SAS: 491, Size 19.86: R143, N114, E110, S139, Q135, A131
SAS: 482, Size 15.76: R164, N167, E134, S139, Q135, A131
SAS: 465, Size 22.69: R143, N115, E110, S139, Q135, A131
SAS: 462, Size 18.17: R143, N138, E134, S139, Q135, A131
SAS: 439, Size 18.17: R143, N138, E110, S139, Q135, A131
Epi#28
SAS: 445, Size 22.79: V50, Q107, W111, E110, Q135, S139, R143
SAS: 426, Size 19.06: V50, Q107, F49, E53, Q57, G46, R44
SAS: 370, Size 19.06: V50, Q107, E110, W111, F49, G46, R44
Epi#31
SAS: 347, Size 21.62: L256, R180, N178, R10, W6, V197, D175
SAS: 339, Size 17.74: L251, R180, N178, R10, W6, V197, D175
Epi#33
SAS: 368, Size 15.95: Q135, Y165, P129, S126, R164
Epi#34
SAS: 445, Size 18.39: V238, W235, S236, G144, R143, S139, S142
SAS: 436, Size 14.07: V238, W235, S236, G144, R143, S142, T141
SAS: 358, Size 18.39: V238, W235, S236, G144, R143, T141, S139
Epi#37
SAS: 415, Size 23.03: T254, A188, L256, R180, N177
SAS: 374, Size 18.04: T254, A188, L256, R180, N179
SAS: 341, Size 19.93: T254, A188, L256, R180, N178
Epi#39
SAS: 323, Size 11.55: A15, E265, H17, R19, P14, G20, L21
SAS: 238, Size 12.13: A15, E265, H17, T22, P14, G20, L21
Epi#40
SAS: 370, Size 15.73: A166, G189, T254, A188, S158
SAS: 360, Size 15.73: A166, G189, Y186, A188, T254
SAS: 324, Size 17.80: A188, G189, Y186, A156, S182
SAS: 321, Size 23.71: A166, G189, Y186, A156, S182
Epi#41
SAS: 228, Size 19.53: P204, Y208, L211, V197, S210
Epi#42
SAS: 554, Size 16.31: L21, P14, S9, Q12, H17, R19, R269
Epi#44
SAS: 406, Size 15.06: L256, R180, Y186, S158, A188, T254
SAS: 398, Size 18.38: L251, R180, Y186, S158, A188, T254
SAS: 395, Size 12.22: L256, R180, Y257, S250, G252, T254
SAS: 392, Size 15.49: L256, R180, Y186, A188, G189, T254
SAS: 387, Size 12.22: L251, R180, Y257, S250, G252, T254
Epi#46
SAS: 581, Size 12.65: A15, R269, R19, P14, N18, G20
Epi#53
SAS: 297, Size 18.06: W235, S234, Q239, K245
SAS: 283, Size 9.54: W235, S234, Q239, K229
SAS: 250, Size 9.46: W235, S234, Q230, K231
SAS: 249, Size 14.49: W235, S236, Q239, K245
SAS: 247, Size 9.46: W235, P233, Q230, K231
Relase:
Epi#05
SAS: 461, Size 17.25: G158, A189, R165, P128, G126, S129
SAS: 439, Size 17.22: G158, A189, R165, P128, G126, S127
SAS: 436, Size 17.25: G158, A189, S159, P128, G126, S129
SAS: 420, Size 17.25: G158, A189, R165, P130, G126, S129
SAS: 414, Size 17.22: G158, A189, S159, P128, G126, S127
Epi#09
SAS: 510, Size 22.37: T22, G20, R19, A15, R270, A267, T250
SAS: 501, Size 22.37: L21, G20, R19, A15, R270, A267, T250
Epi#10
SAS: 458, Size 17.50: D176, N178, N180, S183, F184, G156, R181
SAS: 424, Size 13.68: D176, N213, N154, S183, F184, G156, R181
SAS: 407, Size 15.87: D176, N213, N154, S155, F184, G156, R181
SAS: 392, Size 16.18: D176, N179, N180, S183, F184, G156, R181
SAS: 362, Size 16.73: D176, N213, N154, A157, F184, G156, R181
Epi#12
SAS: 323, Size 9.38: R45, Y90, E88, N43
SAS: 312, Size 13.53: P128, Y162, E135, P130
SAS: 302, Size 9.46: R165, Y162, E135, P130
SAS: 296, Size 9.46: R165, Y166, E135, P130
SAS: 295, Size 13.19: T255, Y187, E190, S159
Epi#18
SAS: 431, Size 15.20: S251, K246, S260, L257, A189, T255, L252
SAS: 398, Size 14.35: S251, K246, S260, L252, G253, T255, L257
SAS: 378, Size 14.35: S251, K246, S260, L257, G253, T250, L252
Epi#19
SAS: 285, Size 21.53: E111, T142, S237, Q240, R242
SAS: 275, Size 12.58: D119, T142, S237, Q240, R242
Epi#23
SAS: 512, Size 22.29: R45, N43, E88, S24, Q231, P234
SAS: 476, Size 19.71: R144, N115, E111, S140, Q136, A132
SAS: 460, Size 13.83: R165, N168, E135, S131, Q136, A132
SAS: 455, Size 20.11: R144, N139, E135, S131, Q136, A132
SAS: 452, Size 15.83: R165, N168, E135, S140, Q136, A132
Epi#25
SAS: 293, Size 13.93: R45, K27, D119, E88
Epi#28
SAS: 502, Size 19.99: V103, Q108, W112, E111, Q136, S140, R144
SAS: 476, Size 21.74: V51, Q108, F50, E54, Q58, S37, R45
SAS: 472, Size 24.93: V103, Q108, F50, E54, Q58, G47, R45
SAS: 469, Size 23.18: V51, Q108, W112, E111, Q136, S140, R144
SAS: 439, Size 19.16: V51, Q108, F50, E54, Q58, G47, R45
Epi#31
SAS: 354, Size 21.73: L257, R181, N179, R10, W6, V198, D176
SAS: 348, Size 17.85: L252, R181, N179, R10, W6, V198, D176
Epi#33
SAS: 396, Size 22.75: Q201, Y204, P205, S37, R45
SAS: 379, Size 22.75: Q201, Y209, P205, S37, R45
SAS: 357, Size 18.39: H63, Y204, P205, S37, R45
Epi#34
SAS: 466, Size 13.97: V239, W236, S237, G145, R144, S143, T142
SAS: 463, Size 18.37: V239, W236, S237, G145, R144, S140, S143
SAS: 387, Size 18.37: V239, W236, S237, G145, R144, S140, T142
Epi#36
SAS: 206, Size 22.37: T250, A267, A15, G20, T22
Epi#37
SAS: 400, Size 22.59: T255, A189, L257, R181, N178
SAS: 359, Size 17.59: T255, A189, L257, R181, N180
SAS: 334, Size 19.35: T255, A189, L257, R181, N179
Epi#39
SAS: 464, Size 16.36: A167, E135, R165, P128, G126, L125
SAS: 444, Size 16.52: A132, E135, R165, P128, G126, L125
SAS: 441, Size 16.36: A167, E190, R165, P128, G126, L125
SAS: 441, Size 18.98: A189, E190, R165, P128, G126, L125
SAS: 423, Size 16.36: A167, E135, R165, P130, G126, L125
Epi#40
SAS: 324, Size 11.66: A97, G60, T57, P55, S56
SAS: 316, Size 17.09: G158, A189, Y187, A157, S183
SAS: 307, Size 14.92: G158, A157, Y187, A189, T255
SAS: 307, Size 15.34: G99, G60, T57, P55, S56
Epi#41
SAS: 222, Size 19.74: P205, Y209, L212, V198, S211
Epi#42
SAS: 544, Size 16.22: L21, P14, S9, Q12, H17, R19, R270
Epi#44
SAS: 421, Size 14.87: L257, R181, Y187, S159, A189, T255
SAS: 415, Size 18.81: L252, R181, Y187, S159, A189, T255
SAS: 389, Size 22.36: V198, R181, Y187, S159, A189, T255
SAS: 389, Size 21.81: I44, R45, Y90, A48, V51, P52
SAS: 386, Size 19.16: I44, R45, Y90, A48, V51, P55
Epi#46
SAS: 557, Size 14.54: A267, R270, R19, P14, N18, G20
SAS: 553, Size 12.63: A15, R270, R19, P14, N18, G20
SAS: 540, Size 13.10: A267, R270, R19, P14, N18, A15
SAS: 444, Size 14.54: A267, R270, R19, P14, G20, A15
Epi#47
SAS: 627, Size 16.22: A267, R270, A15, R19, L21, N18, P14, S9
SAS: 436, Size 15.11: A267, E266, A15, R19, L21, N18, P14, S9
Epi#51
SAS: 545, Size 21.66: L21, R19, H17, D75, S77, I78, S3, W6
SAS: 485, Size 21.66: L21, R19, H17, D75, Q2, I78, S3, W6
Epi#53
SAS: 328, Size 9.43: W236, S235, Q231, K232
SAS: 316, Size 9.43: W236, P234, Q231, K232
SAS: 301, Size 18.21: W236, S235, Q240, K246
SAS: 246, Size 14.68: W236, S237, Q240, K246
“SAS” is solvent accessible surface.
“Size” is the total suface area of the epitope in Å2.

Example 12

The object of this example is to provide evidence showing that subtilisins with an homology to BPN′ of as low as 44,8% reveal a similar epitope distribution as BPN′.

Alcalase, Protease B, Savinase, Esperase, and PD498 (which range from 44.8% to 69.5% in sequence identity to BPN′) were epitope mapped as described in the above example, and compared with epitope mapped BPN′ (FIG. 1).

The data in FIG. 1 show a significant overlap between the areas on the primary structure of the respective proteases. Overall, 6 regions were identified: 1-20, 35-65, 95-115, 130-145, 170-220, and 260-270.

Even better overlap between the epitope sequences can be found among proteins of higher sequence identity, such as within the Savinase-like subtilisins with more than 81% identity, preferably more than 85%, more preferably more than 90%, even more preferably more than 96% or most preferably more than 98% identity.

Example 13 Wash Performance

The following example provides results from a number of washing tests that were conducted under the conditions indicated

TABLE 9
Experimental conditions for evaluation of Subtilisin variants I44V.

	Detergent	OMO Acao
	Detergent dose	2.5 g/l
	PH	10.5
	Wash time	14 min.
	Temperature	25° C.
	Water hardness	9°dH
	Enzymes	Subtilisin variant I44V
	Enzyme conc.	10 nM
	Test system	150 ml glass beakers with a stirring rod
	Textile/volume	5 textile pieces (Ø 2.5 cm) in 50 ml detergent
	Test material	EMPA117 from Center for Testmaterials,
		Holland

TABLE 10
Experimental conditions for evaluation of Subtilisin variants Q12D.

	Detergent	Persil Powder
	Detergent dose	4 g/l
	PH	10.5
	Wash time	20 min.
	Temperature	30° C.
	Water hardness	18°dH
	Enzymes	Subtilisin variant Q12D
	Enzyme conc.	10 nM
	Test system	150 ml glass beakers with a stirring rod
	Textile/volume	5 textile pieces (Ø 2.5 cm) in 50 ml detergent
	Test material	EMPA116 from Center for Testmaterials,
		Holland

TABLE 11
Experimental conditions for evaluation of Subtilisin variants Q12D.

	Detergent	Tide
	Detergent dose	1 g/l
	PH	10.5
	Wash time	10 min.
	Temperature	25° C.
	Water hardness	6°dH
	Enzymes	Subtilisin variant Q12D
	Enzyme conc.	10 nM
	Test system	150 ml glass beakers with a stirring rod
	Textile/volume	5 textile pieces (Ø 2.5 cm) in 50 ml detergent
	Test material	EMPA117 from Center for Testmaterials,
		Holland

pH is adjusted to 10.5 which is within the normal range for a powder detergent.

Water hardness was adjusted by adding CaCl₂ and MgCl₂ (Ca²⁺:Mg²⁺=2:1) to deionized water (see also Surfactants in Consumer Products—Theory, Technology and Application, Springer Verlag 1986). pH of the detergent solution was adjusted to pH 10.5 by addition of HCl.

Measurement of reflectance (R) on the test material was done at 460 nm using a Macbeth ColorEye 7000 photometer. The measurements were done according to the manufacturers protocol.

The wash performance of the variants was evaluated by calculating a performance factor:
P=(R_Variant−R_Blank)/(R_Savinase−R_Blank)

• P: Performance factor
• R_Variant: Reflectance of test material washed with variant
• R_Savinase: Reflectance of test material washed with Savinase®
• R_Blank: Reflectance of test material washed with no enzyme

The variants all have improved wash performance compared to Savinase®—i.e. P>1.

The variants can be divided into improvement classes designated with capital letters:

• Class A: 1<P≦1.5
• Class B: 1.5<P≦2
• Class C: P>2

TABLE 12
Subtilisin variants and improvement classes.

	Improvement class	Variants
	C	I44V, Q12D

As it can be seen from Table 12 SAVINASE® variants of the invention exhibits an improvement in wash performance.