AU2001254623A1

AU2001254623A1 - Production and use of protein variants having modified immunogenicity

Info

Publication number: AU2001254623A1
Application number: AU2001254623A
Authority: AU
Inventors: Steffen Ernst; Esben Peter Friis; Erwin Ludo Roggen; Allan Svendsen; Claus Von Der Osten
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2000-04-28
Filing date: 2001-04-30
Publication date: 2001-11-12
Also published as: AU2001254620A1; AU2001254622A1; EP2258853A1; US20020192792A1; WO2001083770A2; ATE471377T1; US20150037872A1; US20030144165A1; WO2001083761A1; DE60142415D1; WO2001083559A2; EP1280919A2; US20140011259A1; EP2236611A1; ES2588756T3; EP2258835A1; EP1555322B1; EP2258853B1; CA2406621A1; US7226770B2

Abstract

The properties of a fungal lipolytic enzyme can be altered by substituting amino acid residues corresponding to certain specified amino acid residues in the T. lanuginosus lipase. The altered property may be, e.g., an increased thermostability, an altered pH dependence, or an altered substrate specificity.

Description

PROTEIN VARIANTS HAVING MODIFIED IMMUNOGENICITY

Field of invention

The present inventio 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, ppl-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 (Thl) pathway (Int. Arch. Allergy Immunol., 1999, vol. 119, ppl- 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 se- 5 lect the appropriate treatment, including allergen vaccination.

In WO99/53038 (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.

10 599-605) , 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, is 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

20 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) .

25

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 bind- 30 ing 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 O92/10755 a method for modifying proteins to obtain less immunogenic variants is described. Randomly constructed protein variants, revealing a reduced binding of antibodies to the par- ent 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 aicds 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 informa- tion, the results obtained for one protein can not be applied on another protein.

WO 99/47680 (ALK-ABELLO) discloses the identification and modi- fication 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 A 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 com- prising 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, New York (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 Hy- bridization (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 mole- cules 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 (de- oxyadenosine, 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 in- eludes double- tranded 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 se- s quences. 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 .

o 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 rep- 5 lication, 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.

0 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. 5

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 lar- 0 ger 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.

s "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. 0

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 cod- s ing 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 0 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 5 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 0 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 forreign 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 nuceic 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, prefera- bly 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, se isynthetic, syn- thetic 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, syn- thetic 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 poly- peptide 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 US 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 5 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 o 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.

s 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 0 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 termi- 5 nator 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 o 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 pro- teinase 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 in- vention. 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 nu- cleic 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) , Sac- charomyces cerevisiae heme activator protein 1 (hapl) , 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 (Hartl 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 en- coding a chaperone may be obtained from the genes encoding Bacillus subtilis GroE proteins, Aspergillus oryzae protein disul- phide isomerase, Saccharomyces cerevisiae calnexin, Saccharomyces cerevisiae BiP/GRP78, and Saccharomyces cerevisiae Hsp70. 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, Proceed- ings 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 ex2 , 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 oryzae glu- coamylase 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 dihy- drofolate 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 Strepto yces coelicolor agarase gene (dagA) , the Bacillus subtilis levansucrase gene (sacB) , the Bacillus subtilis alkaline protease gene, the Bacillus licheni- formis alpha-amylase gene (amyL) , the Bacillus stearothermophi- lus maltogenic amylase gene (amyM) , the Bacillus amyloliquefa- ciens 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 pro- karyotic 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 promot- ers 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 mie- hei aspartic proteinase, Aspergillus niger neutral al- pha-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 nidu- lans acetamidase, Fusarium oxysporum trypsin-like protease (as described in U.S. Patent 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 (-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 (US 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 Ro anos 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 (Subra ani et al . , Mol. Cell Biol. 1 (1981), 854 -864), the MT-1 (metallothionein gene) promoter (Pal iter 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 (US 4,745,051; Vasuvedan et al . , FEBS Lett. 311, (1992) 7 - 11), the P10 promoter (J.M. Vlak et al . , J. Gen. Virology 69, 1988, pp. 765-776), the Autographa califomica polyhedrosis virus basic protein promoter (EP 397 485) , the baculovirus immediate early gene 1 promoter (US 5,155,037; US 5,162,222), or the baculovirus 39K delayed-early gene promoter (US 5,155,037; US 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 anthrani- late synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease, for fungal hosts) the TPI1 (Al- ber 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 de- scribed 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 mal- togenic amylase gene from Bacillus NCIB 11837, the Bacillus stearothermophilus alpha-amylase gene, the Bacillus licheni- formis 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 (-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 a-factor and Saccharomy- ces 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. US 4,870,008), the signal peptide of mouse salivary amylase (cf. 0. Hagenbuchle et al . , Nature 289,

1981, pp. 643-646), a modified carboxypeptidase signal peptide

(cf. L.A. Vails 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 . , Yeast 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 poly- peptide 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 pep- tide may be the yeast a-factor leader (the use of which is described in e.g. US 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 lepi- dopteran Manduca sexta adipokinetic hormone precursor signal peptide (cf. US 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 re- striction 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 appro- priate 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 ex- trachromosomal 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, chlorampheni- col, tetracycline, neomycin, hygromycin or methotrexate resis- tance. 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 (acetami- dase) , argB (ornithine carbamoyltransferase) , bar (phosphi- nothricin acetyltransferase) , hygB (hygromycin phosphotrans- ferase) , niaD (nitrate reductase), pyrG (orotidine-5' -phosphate decarboxylase) , sC (sulfate adenyltransferase) , trpC (anthrani- late 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 non- homologous 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 probabil- ity 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, pUBHO, pE194, pTA1060, and pAMβl . 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 poly- peptide 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 se- lecting 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 poly- peptides. 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 ex- tra-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 al- kalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringien- sis; 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., Shi- gekawa and Dower, 1988, Biotechniques 6:742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriol- ogy 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 in- troduced in the cells are described in e.g. Kaufman and Sharp,

J. Mol. Biol. 159 (1982), 601 - 621; Southern and Berg, J. Mol.

Appl. 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, Basidiomy- cota, 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 (Endo y- cetales) , basidiosporogenous yeast, and yeast belonging to the Fungi Imperfect! (Blastomycetes) . The ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomy- cetaceae. The latter is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces) , Nad- sonioideae, 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 in- vention, 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, Hansehula, , or Yarrowia. In a preferred embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomy- ces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces ovifor is cell. Other useful yeast host cells are a Kluyveromyces lactis Kluyveromyces fragilis Hansehula po- lymorpha, Pichia pastoris Yarrowia lipolytica, Schizosaccharo- myces pombe, Ustilgo maylis, Candida maltose, Pichia guiller- mondii and Pichia methanolio cell (cf. Gleeson et al . , J. Gen. Microbiol. 132, 1986, pp. 3459-3465; US 4,882,279 and US 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, Penicil- lium, 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 Neu- rospora 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, Aspergil- lus 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 tricho- thecioides cell. In another prefered embodiment, the filamen- tous fungal parent cell is a Fusarium strain of the section Ele- gans, e.g., Fusarium oxysporum. In another most preferred embodiment, the filamentous fungal host cell is a Humicola inso- lens 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 pur- purogenum cell. In another most preferred embodiment, the filamentous fungal host cell is a Thielavia terrestris cell or a 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 de- scribed in EP 238 023 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 Ma- lardier et al . , 1989, Gene 78:147-156 or in copending US Serial 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, EP 184 ... The transformation of F. oxysporum may, for in- stance, 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 Enzy- mology, 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 US 4,745,051; US 4, 775, 624; US 4,879,236; US 5,155,037; US 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. US 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 centrifu- gation 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 chroma- tography, 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 in- eluding 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 <5A 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 epi- tope, 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.

Detailed description of the invention

comprising the steps of:

a) obtaining antibody binding peptide sequences,

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

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 iso- late 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 IgE 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 calcu- lated 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 se- quences 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 Giege: 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 ho- mologous 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 sur- face 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 <5A, preferably <3A 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 se- quences, 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 pro- tein.

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 5A 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.

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 pro- tein.

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, inser- tion, deletion will be indicated by: "Posi tion 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 5 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, o 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 resi- 5 dues 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.

0

Chemical conjugation

For chemical conjugation, the protein variant needs to be incubate with an active or activated polymer and subsequently sepa- 5 rated 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.

o 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, di- vinylsulfone, 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/a ide (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 (US patent 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. A r. 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 nucleo- philes 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 nondestructive 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 (US patent no. 5,122,614, (1992); Zalipsky et al., (1992), Biotechnol. Appl. Biochem., 15, p. 100-114; Mon- fardini 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 Al, (1993), Looze, Y.) etc. The derivatives are usually made by reacting the chlorofor- mate 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 (US patent 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 (US patent 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; US patent 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 (US patent 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 l,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 W096/17929 and WO99/00489 (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 acti- vated 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 modi- fications 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 ex- pressed 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 glycosyla- tion, phosphorylation, N-terminal processing, acylation, ribosy- lation 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 5 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 spe- o cific 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 phophoryla- tion sites with the recognition sequence arg-arg- (xaa)_n-ser s (where n = 0, 1, or 2) , 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, which can usually be phophorylated by tyrosine-specific kinases (T.E. Creighton, "Proteins- Structures and molecular proper- 0 ties", 2nd ed. , Freeman, NY, 1993).

Randomized approaches to introduce modifications in epitope areas . 5

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 func- o tionality 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.

5 A diversified library can be established by a range of techniques known to the person skilled in the art (Reetz MT; Jaeger KE, in 'Biocatalysis - from Discovery to Application' edited by Fessner WD, Vol. 200, pp. 31-57 (1999); Stemmer, Nature, vol. 370, p.389-391, 1994; Zhao and Arnold, Proc. Natl. Acad. Sci., 0 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 earring out PCR mutagenesis using one or more oligonucleotide primers which s 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 0 predetermined distribution function. Algorithms have been disclosed, which facilitate this design (Jensen LJ et al . , Nucleic Acids Research, Vol. 26(3), 697-702 (1998)).

In an embodiment substitutions are found by a method comprising 5 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 se- o quence 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 Biotechnol- ogy, vol. 17, pp. 893-896, 1999) and/or combination with other methods of reducing immunogenicity by genetic means (such as that disclosed in WO92/10755) .

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 to- wards 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 (Stem- mer, Nature 370, pp. 389-391, 1994 and US 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 US 5,830,721. The single- stranded oligonucleotides could be par- tially 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 cor- responding 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 seg- ments, 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 vi- tro assays for immunogenicity such as assays based on cytokine expression profiles or other proliferation or differentiation responses of epithelial and other cells incl . 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 litera- ture (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-ABELLO) . Also, sev- 5 eral cellular assays could be employed to show the modified im- uneresponse 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", Immu- 0 nol Today, 1996, vol. 17, pp. 526-532; Hoffmann et al . , Allergy, 1999, vol. 54, pp. 446-454 , -WO99/07880) .

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

Determining functionality 0

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 (WO99/34011, Genencor S International; J.E. Ness, et al, Nature Biotechn., 17_, pp. 893- 896, 1999), oxidoreductase assays (Cherry et al . , Nature Biotechn., 37 , pp. 379-384, 1999, and assays for several other enzymes (W099/45143, Novo Nordisk). Those assays that employ soluble substrates can be employed for direct analysis of function- 0 ality 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 5_0_^, 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 cre e, hair dye, toothpaste, household articles, agro chemicals, personal care products, such as cleaning preparations e.g. for contact lenses, cos- metics, 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, carbohy- drase, 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 pro- duced, e.g., as disclosed in US 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 ethox- ysulfate (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 mono- ethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as de- scribed 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 com- plexing agent such as zeolite, diphosphate, triphosphate, phos- phonate, citrate, nitrilotriacetic acid (NTA) , ethylene- diaminetetraacetic 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) , poly- ethyleneglycol (PEG), poly (vinyl alcohol) (PVA) , polycarboxylates such as polyacrylates, maleic/acrylic 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 tetraace- tylethylenediamine (TAED) or nonanoyloxybenzenesulfon-ate (NOBS) . Alternatively, the bleaching system may comprise peroxyacids of, e.g., the amide, i ide, 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, bacte- ricides, 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 espe- cially alkali metal pyrophosphates, orthophosphates, and po- lyphosphates . An example of phosphorus-containing organic alkaline detergent builder, when present, includes the water-soluble salts of phosphonates . Examples of non-phosphorus-containing in- organic 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, malo- nates, fatty acid sulphonates, carboxymetoxy succinates, ammonium polyacetates, carboxylates, polycarboxylates, amino- polycarboxylates, polyacetyl carboxylates and polyhydroxsul- phonates .

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 poly- acrylic/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 per- phosphates . Preferred activator materials are TAED and glycerol triacetate.

The dishwashing detergent composition of the invention may be stabilized using conventional stabilizing agents for the en- zyme(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. defloc- culant 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, US 5141664, EP 561452, EP 561446, GB 2234980, WO 93/03129, EP 481547, EP 530870, EP 533239, EP 554943, EP 346137, US 5112518, EP 318204, EP 318279, EP 271155,

EP 271156, EP 346136, GB 2228945, CA 2006687, WO 93/25651, EP 530635, EP 414197, US 5240632. 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 lo- tion, 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 con- tact 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 prod- ucts, 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 surf ce . Oxidoreductases

The most common oxidoreductase for personal care purposes is an oxidase (usually glucose oxidase) with substrate (e.g. glucose) that ensures production of H₂0₂, which then will initiate the oxidation of for instance SCN^" or I^" into antimicrobial reagents (SCNO^" or I₂) by a peroxidase (usually lactoperoxidase) . This en- zymatic 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 -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 prote- ase .

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 doug .

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 un- malted 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, li- poxygenase, 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 dex- trins that result in gumminess in the finished product . The en- zyme 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.

The bread volume can be improved by fungal α-amylases which fur- ther 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 ther olabile 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. Pen- tosanases 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 α-amylases. Malto- dextrins, primary consisting of oligosaccharides and dextrins, are obtained.

The mixture is then treated with an amyloglucosidase for hydro- lysing the oligosaccharides and dextrins into glucose. This way a sweeter product is obtained. If high maltose syrups are desired β-amylases alone or in combination with a pullulanase (de-branch- ing 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 α-amylases is advantageously being used for thinning of starch in distilling mashes.

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

In the dairy industry β-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) .

α-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 US patent 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 Nord

Phytases

Phytases of the invention may advantageously be used in the manu- facturing 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 US patent no. 3,297,548) .

5 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 10 358) .

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) .

15

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

Textile applications

20

Proteases

Proteases are used for degumming and sand washing of silk.

25 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) .

30

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 con- siderable 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 of- fers 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 anti- body 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 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 afore- mentioned 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 polypep- tides, 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 go- nadotropin, 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 phar- maceuticals.

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 ef- feet, 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 aspa- ratate. 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 mem- brane, 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; Ce- cropin A, B and PI; 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, Droso ycin, gl-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 Cen- traallbureau 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 Aspergil - lus 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 (Pro- lyl 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 (Tryp- tophanyl 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 (Cari- cain) 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, NY, pp. 271-272) .

The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Dalton range. They are inhibited by diisopro- pylfluorophosphate . 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 mesenteri- copeptidase, 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™, 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, Rus- 5 sel et al., (1987), Nature, 328, p. 496-500, WO 88/08028 (Genex), WO 88/08033 (A gen) , WO 89/06279 (Novo Nordisk A/S) , WO 91/00345 (Novo Nordisk A/S) , EP 525 610 (Solvay) and WO 94/02618 (Gist- Brocades N.V. ) .

lo The activity of proteases can be determined as described in "Methods of Enzymatic Analysis", third edition, 1984, Verlag Che- ie, Weinheim, vol. 5.

is 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

20 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 Al, EC 3.1.1.73 feruloyl esterase or EC 3.1.1.74 cutinase.

25 The parent lipolytic enzyme may be prokaryotic, particularly a bacterial enzyme, e.g. from Pseudomonas . Examples are Pseudomo- nas lipases, e.g. from P. cepacia (US 5,290,694, pdb file 10IL) , P. glumae (N Frenken et al . (1992), Appl. Envir. Microbiol . 58 3787-3791, pdb files 1TAH and 1QGE) , P. pseudoalcaligenes (EP

30 334 462) 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 Chro- mobacteriu viscosum (DE 3908131 Al) . Other examples are bacte- rial cutinases, e.g. from Pseudomonas such as P. mendocina (US 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 so- lani pisi (S. Longhi et al . , Journal of Molecular Biology, 268 (4), 779-799 (1997)) and Humicola insolens (US 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 US 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. lanugino- sus, 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. het- erosporum (R87979) , lysophospholipase from Aspergillus foetidus (W33009) , phospholipase Al from A. oryzae (JP-A 10-155493) , li- pase from A. oryzae (D85895) , lipase/ferulic acid esterase from A. niger (Y09330) , lipase/ferulic acid esterase from A. tubin- gensis (Y09331) , lipase from A. tubingensis (WO 98/45453) , lysophospholipase from A. niger (WO 98/31790) , lipase from F. so- lanii having an isoelectric point of 6.9 and an apparent molecu- lar 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 in- corporated 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 (US patent no. 4,950,417 (Solvay enzymes)) or Ps. alcaligenes and Ps . pseudoalcaligenes (EP 218 272) 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 (Kugi iya 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. pu ilus (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 en- zymes . 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 Che- mie, 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 _NAD (P)⁺_ (1.4.1.3), Glutamate dehydrogenase _NADP⁺_ (1.4.1.4), L- A ino 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), Lipoxy- genase (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, My- celiophtora thermophila, Coprinus cinereus, Rhizoctonia solani, Rhizoctonia praticola, Scytalidium thermophilum and Rhus ver- nicifera. 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 "Copri - nus-like laccase" is intended to indicate a laccse 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 Coprinus 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 DK patent applications No. 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 re- ductases 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 Che- mie, 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 in- eluded 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:

α-amylase (3.2.1.1) β-amylase (3.2.1.2), glucan 1,4-α- glucosidase (3.2.1.3), cellulase (3.2.1.4), endo-1, 3 (4) -β- glucanase (3.2.1.6), endo-1, 4-β-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), β-glucosidase (3.2.1.21), α-galactosidase (3.2.1.22), β-galactosidase (3.2.1.23), amylo-1, 6-glucosidase (3.2.1.33), xylan 1, 4-β-xylosidase (3.2.1.37), glucan endo-1, 3-β- D-glucosidase (3.2.1.39), α-dextrin endo-1, 6-glucosidase (3.2.1.41), sucrose α-glucosidase (3.2.1.48), glucan endo-1, 3 -α- glucosidase (3.2.1.59), glucan 1, 4-β-glucosidase (3.2.1.74), glucan endo-1, 6-β-glucosidase (3.2.1.75), arabinan endo-1, 5-α- 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 α-1, 3-glucanases derived from Trichoderma harzianum; α-1, 6-glucanases derived from a strain of Paecilomyces; β-glucanases derived from Bacillus subtilis; β-glucanases derived from Humicola insolens; β-glucan-ases derived from Aspergillus niger; β-glucanases derived from a strain of Trichoderma; β-glucanases derived from a strain of Oerskovia xanthineolytica; exo-1, 4-α-D-glucosidases (glucoamy- lases) derived from Aspergillus niger; α-amylases derived from Bacillus subtilis; α-amylases derived from Bacillus amyloliquefa- ciens; α-amylases derived from Bacillus stearothermophilus; α- amylases derived from Aspergillus oryzae; α-amylases derived from non-pathogenic microorganisms; α-galactosidases derived from Aspergillus niger; Pentosanases, xylanases, cellobiases, cellu- lases, hemi-cellulases deriver from Humicola insolens; eellulases derived from Trichoderma reesei; eellulases derived from non- pathogenic mold; pectinases, eellulases, arabinases, hemi- celluloses derived from Aspergillus niger; dextranases derived from Penicillium lilacinum; endo-glucanase derived from non- pathogenic mold; pullulanases derived from Bacillus acidopullyti- cus; β-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^®, Cere yl^®, Citrozym™, Denimax™, Dezyme™, Dextrozyme™, Finizym^®, Fungamyl™, Gamanase™, Glucanex^®, Lac- tozym^®, Maltogenase™, Pentopan™, Pectinex™, Promozyme^®, Pulp- zye™, Novamyl™, Terma yl^®, 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 eellulases, e.g. Pseudomonas eellulases or Bacillus, such as the Bacillus strains described in US 4,822,516, US 5,045,464 or EP 468 464, or B. lautus (cf. WO 91/10732), eellulases. More preferably, the parent eellulases may be a fungal cellulase, in particular Humicola, Trichoderma, Irpex, Aspergillus, Penicillium, Myceliophthora or Fusarium eellulases. Examples of suitable parent eellulases are described in, e.g. WO 91/17244. Examples of suitable Trichoderma eellulases are those described in T.T. Teeri, Gene 51, 1987, pp. 43-52. Preferably, the parent cellulase is selected from the eellulases 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 α-amylase

It is well known that a number of α-amylases produced by Bacillus spp. are highly homologous on the amino acid level. For instance, the B . licheniformis α-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 . amyloliguefaciens α-amylase comprising the amino acid sequence shown in SEQ ID NO: 5 of WO 00/29560 and about 79% homologous with the B. stearothermophilus α-amylase comprising the amino acid sequence shown in SEQ ID NO: 3 of WO 00/29560. Further homologous α-amylases include an α-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 α-amylase described by Tsukamoto et al . , Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31.

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

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

Accordingly, in the present context, the term "Termamyl-like α- amylase" is intended to indicate an α-amylase which, at the amino acid level, exhibits a substantial homology to Termamyl™, i . e . , the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID NO: 4 (WO 00/29560) . In other words, a Termamyl- like α-amylase is an α-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 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 : or 2 or 3 or 4 or 5 or 6 or 7 or 8 of WO 00/29560 and/or ii) displays immunological cross-reactivity with an antibody raised against one or more of said α-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 α- 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 eodon 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, Wisconsin, USA 53711) .

The parent Termamyl-like α-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 α-amylase may be used to identify equivalent/corresponding positions in other Termamyl-like α- 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 Gl and G2 disclosed in Boel et al . (1984), "Glucoamylases Gl 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 Gl 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, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (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 Gl 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 Che- ie, 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 transgluta- minase 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 pep- tide-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 A/S) .

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 Bacteriol- ogy, 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., US 5,156,956; Andou et al . , US 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 . , US 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 ficuu , Aspergillus awamori, Aspergillus oryzae, Aspergillus terreus or Aspergillus nidulans, and various other Asper- gillus 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 : Peetate 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 PDIs described in WO 95/01425 (Novo Nordisk A/S) and suitable glucose isomerases include those described in Bio- technology 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 Urti- cales; including those from Betula, Alnus , Corylus, Carpinus, Olea, Phleum pratense and Artemisia vulgaris, such as Aln gl, Cor al, Car bl, Cry jl, Amb al and a2 , Art vl, Par jl, Ole el, Ave vl, and Bet vl (WO 99/47680) .

Mite allergens include but are not limited to those from Derm. farinae and Derm, pteronys., such as Der fl and f2 , and Der pi 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 dl; Can fl; Equ cl ; Equ c2 ; Equ c3.

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

Fungal allergens include those from Alternaria alt. and Cladospo. herb, such as Alt al and Cla hi.

Food allergens include but are not limited to those from milk (lactoglobulin) , egg (ovalbumin) , peanuts, hazelnuts, wheat (alfa-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) ) NaCI 8.00 g

KCl 0.20 g

K₂HPO₄ 1.04 g

KH₂P0₄ 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 0.1M, 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-AAPF-pNP) Sigma no. S-7388, Mw 624.6 g/mol .

- mPEG (Fluka)

Colouring 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 Appen- dix 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 og 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, KLASD, KLYSD, KLY-D, R-M-D. The epitope searching algorithm

The "core" of the program is the algorithm that scans the pro- 5 tein surface for the epitope patterns. The principle is that several "trees" are built, where each of their branches describes one epitope:

l.All residues in the protein are checked according to: a) o 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 usu- 5 ally 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 0 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. 5 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 o 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 con- sensus 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 ulti- plied 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 algo- rithm 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 there- fore 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 con- sensus 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 structure/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 calcu- lated) . 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 epi- topes 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 figure 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) NaCI (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) NaCI (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 Bo - holdtgaard, 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 IgG:

Specific IgGl and IgE levels were determined using the ELISA specific for mouse or rat IgGl 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 concen- tration 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 200x 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 3x with 300 microliter washing buffer.

Unknown rat sera and a known rat IgE solution were diluted in dilution buffer: Typically lOx, 2Ox and 4Ox for the unknown sera, and % 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 3x with washing buffer. The anti-rat IgE (biotin) was diluted 2000x in dilution buffer. 100 microliter was added to each well. Incubation was for 1 hour at room temperature. Unbound material was removed by washing 3x with washing buffer.

Streptavidin was diluted lOOOx in dilution buffer. 100 microli- ter was added to each well . Incubation was for 1 hour at room temperature. Unbound material was removed by washing 3x with 300 microliter washing buffer. OPD (0.6 mg/ml) and H₂0₂ (0.4 micro- liter /ml) were dissolved in citrate buffer. 100 microliter was added to each well. Incubation was for 30 minutes at room tem- perature. The reaction was stopped by addition of 100 microliter H₂S0₄. The plates were read at 492 nm with 620 n as reference. Similar determination of IgG can be performed using anti Rat-IgG and standard rat IgG 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 ahti-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, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (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, Savi- nase.

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 AQTVPYGIPLIKADKVQAQGFKGAV VAVLDTGIQASHPDL WGGASFVAGEAYN-TD

BPN', 1 AQSVPYGVSQIKAPALHSQGYTGSNVVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ** **** *** ** * ****** * ** ***** * **** * * * *

Alcalase, 60 GNGHGTHVAGTVAADNTTGV GVAPSVSLYAVV NSSGSGSYSGIVSGIE ATTNGMD BPN ' 61 DNSHGTHVAGTVAA WSIGVLGVAPSSALYAVKVLGDAGSGQYS IINGIEAIANMD * ************ * ******** ******* *** ** * ***** * **

Alcalase, 120 VINMSLGGASGSTAMKQAVDNAYARGWWAAAGNSGSSGNTNTIGYPA YDSVIAVGAV

BPN' , 121 VINMSLGGPSGSAAKAAVDKAVASGWWAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV

******** *** * * *** * * ********** ** * * *** ** ********

Alcalase, 180 DSNSNRASFSSVGAE EVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALI SKHPN BP ' , 181 DSSNQRASFSSVGPE D APGVSIQST PGNKYGAYNGTSMASPHVAGAAALI SKHPN

10 ** ******** ** ***** ** * * * ***********************

Alcalase, 240 LSASQVRNRLSSTATY GSSFYYGKGLINVEAAAQ BPN' , 2 1 WTNTQVRSS QNTTTKLGDSFYYGKGLINVQAAAQ

*** * * * ** *********** ****

15

Protease B:

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

20 PROTEASE B, 1 AQTIPWGISRVQAPAAHNRGLTGSGVKVAV DTGI-STHPDLNIRGGASFVPGE-PSTQD

BPN', 1 AQSVPYGVSQIKAPAHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD

** * * * *** * * *** ***** * ** * **** **** ** * * **

PROTEASE B, 59 GNGHGTHVAGTIAANNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLE AGNNGMH 25 BPN¹ , 61 DNSHGTHVAGTVAANNSIGVLGVAPSSALYAVKVLGDAGSGQYS IINGIE AIANN D

* ******** *************** ******** *** * * * *** * *

PROTEASE B, 119 VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSG GSISYPARYANAAVGAT

BPN', 121 VINMS GGPSGSAAΓJKAAVDKAVASGV WAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV

30 * * *** ** ** * ** * ** **** ** * ** * ****

PROTEASE B, 175 DQNNNRASFSQYGAGLDIMAPGVNIQSTYPGSTYASDNGTSMATPHVAGAAALVKQKNPS BPN', 181 DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN * * ***** * ** ***** **** ** * ****** ********* * *

35

PROTEASE B, 235 WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR BPN', 241 WTNTQVRSSLQNTTTK GDSFYYGKGLINVQAAAQ

* * * * * ** * ** ** ** * **

40

Esperasβ:

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

45 Esperase, 1 QTVP GISFINTQQAHNRGIFGNGARVAVLDTGI-ASHPDLRIAGGASFISSE-PSYHDN BPN', 2 QSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPD KVAGGASMVPSETPNFQDD

* ** * * * * * * *** * ** ***** ***** ** * *

Esperase, 59 NGHGTHVAGTIAANNSIGV GVAPSADLYAVKV DRNGSGS ASVAQGIEAINNN HI 50 BPN', 62 NSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMDV

* ******** *************** ******* *** ****** ***

Esperase, 119 INMSLGSTSGSSTLE AVNRANNAGILVGAAGNTGRQG VNYPARYSGVMAVAAVD

BPN' , 122 INMSLGGPSGSAA AAVDKAVASG AAAGNEGSTGSSSTVGYPGKYPSVIAVGAVD

55 ****** *** * ** * * * **** * * * ** * * ** ***

Esperase, 175 QNGQRASFSTYGPEIElSAPGVNVNSTYTGNRYVS SGTSMATPHVAGVAALVKSRYPS BPN¹ , 182 SSNQRASFSSVGPELDVMAPGVSIQST PGNKYGAYNGTSMASPHVAGAAA ILSKHPNW ****** *** **** ** ** * ***** ***** *** * *

60

Esperase, 235 TNNQIRQRINQTATYLGSPSLYGNGLVHAGRATQ BPN' , 2 2 TNTQVRSSLQNTTTKGDSFYYGKGLINVQAAAQ

** * * * * ** ** ** * *

Protease C:

65 59.6% identity in 275 residues overlap; Score: 825.0; Gap frequency: 2.2%

ProteaseC, 1 AQSVPWGISRVQAPAAHNRGLTGSGVRVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN' 1 AQSVPYGVSQI APALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** * * *** * * *** * *** * ** * **** **** ** * * **

ProteaseC, 59 GNGHGTHVAGTIAALNNSIGVLGVAPSAE YAVKVLGASGSGSYSSIAQGLEWAGNNGH BPN' , 61 DNSHGTHVAGTVAALNNSIGV GVAPSSALYAVKVLGDAGSGQYS IINGIEWAIANNMD

* ******** *************** ******** *** ** * * *** * *

ProteaseC, 119 VASLSLGSPSPSATLEQAVNSATSRGVLWAASGNSGA GSISYPARYANAMAVGAT

BPN¹, 121 VINMSLGGPSGSAALKAAVDKAVASGWVAAAGNEGSTGSSSTVGYPG YPSVIAVGAV * *** ** ** * ** * ** **** ** * ** * ****

ProteaseC, 175 DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAAVKQKNPS BPN' , 181 DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALI SKHPN

* * ***** * ** **** *** ** * ****** ********* * *

ProteaseC, 235 SNVQIRNHLKNTATSLGSTNLYGSGLVNAEAAAR BPN' , 241 WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ

* * * * * ** * ** ** ** * ***

Protease D:

59 .3% identity in 275 residues overlap ; Score : 815 . 0 ; Gap frequency: 2 . 2% ProteaseD, 1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAV DTGI-STHPDLNIRGGASFVPGE-PSTQD

BPN ' , 1 AQSVPYGVSQIKAPAIIHSQGYTGSNVKVAVIDSGIDSSHPDIJKVAGGASMVPSETPNFQD

***** * * *** * * *** ***** * ** * **** **** ** * * **

ProteaseD, 59 GNGHGTHVAGTIAALDNSIGVLGVAPSAELYAVKVLGASGSGAISSIAQGLEWAGNNGMH BPN', 61 DNSHGTHVAGTVAALNNSIGVLGVAPSSAYAVKVLGDAGSGQYSWIINGIEAIANND

* ******** *** *********** ******** *** * * * *** * *

ProteaseD, 119 VANLSLGSPSPSATLEQAVNSATSRGVIjVVAASGNSGA GSISYPARYANAMAVGAT

BPN', 121 VINMSLGGPSGSAAIi AAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *** ** ** * ** * ** **** ** * ** * ****

ProteaseD, 175 DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTS ATPHVAGAAAVKQKNPS BPN', 181 DSSNQRASFSSVGPELDV APGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN

* * ***** * ** **** *** ** * ****** ********* * *

ProteaseD, 235 WSNVQIRNH KNTATSLGSTN YGSGLVNAEAATR BPN', 241 TNTQVRSSLQNTTTKLGDSFYYGKG INVQAAAQ

* * * * * ** * ** ** ** * **

Protease E:

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

ProteaseE, 1 AQSVP GISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN', 1 AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD

***** * * *** * * *** ***** * ** * **** **** ** * * **

ProteaseE, 59 GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGGGAISSIAQG EWAGNNGMH BPN', 61 DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAV V GDAGSGQYSWIINGIE AIANNMD

* ******** *************** ******** * * * * * *** * *

ProteaseE, 119 VANLSLGSPSPSAT EQAVNSATSRGVWAASGNSGA DSISYPARYANA AVGAT

BPN', 121 VINMSLGGPSGSAAL AAVD AVASGWWAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV

* * *** ** ** * ** * ** **** ** * ** * ****

ProteaseE, 175 DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAVLVKHKNPS BPN', 181 DSSNQRASFSSVGPELDVMAPGVSIQST PGNKYGAYNGTSMASPHVAGAAALI S HPN

* * ***** * ** **** *** ** * ****** ******* * * * ProteaseE, 235 SNVRIRDHLKKTATS GSTNLYGSGVNAEAATR BPN', 241 WTNTQVRSSLQNTTTKLGDSFYYGKG INVQAAAQ * * * * * * ** ** ** * ** Protease A: 58.9% identity in 275 residues overlap; Score: 812.0; Gap frequency: 2.2%

Protease A, 1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD

BPN', 1 AQSVPYGVSQIKAPALHSQGYTGSNVVAVIDSGIDSSHPDLVAGGASMVPSETPNFQD

***** * * *** * * *** ***** * ** * **** **** ** * * **

Protease A, 59 GNGHGTHVAGTIAANNSIGVLGVAPSAE YAVKVLGASGSGSVSSIAQG EWAGNNGMH BPN', 61 DNSHGTHVAGTVAANNSIGVLGVAPSSAYAVKV GDAGSGQYSWIINGIEWAIANNMD

* ******** *************** ******** *** * * * *** * * Protease A, 119 VANLS GSPSAGGTLEQAVNSATSRGVWAASGNSGA GSISAPASYANAMAVGAT

BPN', 121 VINMSLGGPSGSAA KAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV

* * *** ** * ** * ** **** ** * * * ****

Protease A, 175 DQNNNRASFSQYGPGDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS BPN', 181 DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAA ILSKHPN

* * ***** ** ** **** *** ** * ****** ********* * *

Protease A, 235 WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR BPN', 241 WTNTQVRSSLQNTTTKLGDSFYYG GIilNVQAAAQ * * * * * ** * ** ** ** * **

PD498 :

4 477..77%% i:dentity in 266 residues overlap; Score: 487.0; Gap frequency: 4.9%

PD498, 13 YGPQNTSTPAAWDVTRGSSTQTVAVDSGVDYNHPDLAR VI GYDFIDRDN-NPDLNG

BPN' , 6 YGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDL--KVAGGASMVPSETPNFQDDNS ** ** * *** *** * **** ** * * * *

PD498, 72 HGTHVAGTVAADTNNGIGVAGMAPDTKILAVRVLDANGSGSLDSIASGIRYAADQGAVL

BPN' , 64 HGTHVAGTVAA-LNNSIGVLGVAPSSAYAV VLGDAGSGQYSWIINGIEWAIANNMDVI *********** ** *** * ** ** ** *** * ** * *

PD498, 132 NLS GCECNSTTLKSAVDYANKGAWAAAGND NVSRTFQPASYPNAIAVGAIDS

BPN' , 123 NMSLGGPSGSAALKAAVDKAVASGVWAAAGNEGSTGSSSTVGYPGKYPSVIAVGAVDS

* *** * ** *** * * ******** * * ** ***** **

PD498, 188 NDRKASFSNYGT VDVTAPGVNIASTVPNNGYSYMSGTSMASPHVAGLAAL ASQGKN- -

BPN' , 183 SNQRASFSSVGPELDVMAPGVSIQSTLPGN YGAYNGTSMASPHVAGAAALILSKHPNWT **** * ** **** * ** * * * *********** *** * *

PD498, 246 NVQIRQAIEQTADKISGTGTNF YG

BPN' , 243 NTQVRSSLQNTTTKL---GDSFYYGK

* * * * * * * ***

Properase :

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

Properase, 1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAV DTGI-STHPDLNIRGGASFVPGE-PSTQD BPN', 1 AQSVPYGVSQIKAPALHSQGYTGSNVRVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD

***** * * *** * * *** ***** * ** * **** **** ** * * **

Properase, 59 GNGHGTHVAGTIAALNNSIGVLGVAPNAELYAVKV GASGGGSNSSIAQGLEWAGNNGMH BPN', 61 DNSHGTHVAGTVAANNSIGVLGVAPSSALYAVKV GDAGSGQYS IINGIEAIANNMD

* ******** ************** ******** * * * * * *** * *

Properase, 119 VANLSLGSPSPSATLEQAVNSATSRGVL AASGNSGA GSISYPARYANAMAVGAT

BPN', 121 VINMS GGPSGSAA KAAVDKAVASGWWAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV

* * *** ** ** * ** * ** **** ** * ** * ****

Properase, 175 DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYAS NGTS ATPHVAGAAAV QKNPS BPN', 181 DSSNQRASFSSVGPELDVMAPGVSIQST PGNKYGAYNGTSMASPHVAGAAALILS HPN

* * ***** * ** **** *** ** * ****** ********* * * Properase, 235 WSNVQIRNHLKNTATSLGSTN YGSGLVNAEAATR BPN', 241 TNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ * * * * * ** * ** ** ** * **

Relase:

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

Relase , 1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGIDSTHPD NIRGGASFVPGE-PSTQD

BPN' , 1 AQSVPYGVSQI APALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** * * *** * * *** ***** * **** **** **** ** * * **

Relase, 60 GNGHGTHVAGTIAALDNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLE AGNNGMD

BPN', 61 DNSHGTHVAGTVAALNNSIGV GVAPSSAYAVKVLGDAGSGQYSWIINGIE AIANNMD

* ******** *** *********** ******** *** * * * *** * **

Relase, 120 VAN SLGSPSPSAT EQAVNSATSRGVWAASGNSGA GSISYPARYANAMAVGAT

BPN', 121 VINMSLGGPSGSAALKAADKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV

* * *** ** ** * ** * ** **** ** * ** * **** Relase, 176 DQNNNRASFSQYGAE DIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAAVLQKNPS BPN', 181 DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN

* * ***** * *** **** *** ** * ****** ********* * * * Relase, 236 SNVQIRNHLKNTATSLGSTNLYGSGVNAEAATR BPN', 241 TNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ

* * * * * ** * ** ** ** * **

Savinase:

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

Savinase, 1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN' , 1 AQSVPYGVSQIKAPAHSQGYTGSNV VAVIDSGIDSSHPD KVAGGASMVPSETPNFQD

***** * * *** * * *** ***** * ** * **** **** ** * * ** Savinase, 59 GNGHGTHVAGTIAANNSIGVLGVAPSAE YAVKVLGASGSGSVSSIAQGLE AGNNGMH BPN' , 61 DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKV GDAGSGQYS IINGIE AIANNMD * ******** *************** ******** *** * * * *** * *

Savinase, 119 VANLS GSPSPSATLEQAVNSATSRGVLWAASGNSGA GSISYPARYANAMAVGAT BPN', 121 VINMSLGGPSGSAALKAAVDKAVASGVWVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV

* * *** ** ** * ** * ** **** ** * ** * ****

Savinase, 175 DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS BPN', 181 DSSNQRASFSSVGPELDVMAPGVSIQST PGNKYGAYNGTSMASPHVAGAAALI SKHPN

* * ***** * ** **** *** ** * ****** ********* * *

Savinase, 235 WSNVQIRNHLKNTATSLGSTNYGSGVNAEAATR BPN', 241 WTNTQVRSS QNTTTKLGDSFYYGKG INVQAAAQ

* * * * * ** * ** ** ** * **

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 IB 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 Wiscon- sin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-45) .

The sequence of the YaB protease is disclosed by Kaneko,R.; Ko- yama,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 Yama- gata,Y. ; Isshiki,K.; Ichishima, E. ; Subtilisin Sendai from alka- lophilic 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 IB:

Alignment of YAB protease to BPN': 55,3% identity CLUSTAL (1.7) multiple sequence alignment

YAB -QTVPWGINRVQAPIAQSRGFTGTGVRVAVLDTGISN-HADLRIRGGASFVPGE-PNISD BPN" AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD

*.**.*.₍;_;.** .*.*.**. _t*. ***;*.**, _# * _# ** .. ****.** _^ * **._p*

YAB GNGHGTQVAGTIAALNNSIGVLGVAPNVDLYGVKVLGASGSGSISGIAQGLQWAANNGMH

BPN" DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD _#*_#***;**** ;**************. ** _# ***** _;***_t * * .*..** *.*,

YAB IAN SLGSSAGSATMEQAVNQATASGVLWAASGNSG AGNVGFPARYANAMAVGAT

BPN' VINMSLGGPSGSAALKAAVDKAVASGWWAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV

. *****, _ .***.. Λ*..*_^****;****;**.* . , ,**;*, ;* _{# #} .****_#

YAB DQNNNRATFSQYGAGLDIVAPGVGVQSTVPGNGYASFNGTSMATPHVAGVAALVKQKNPS

BPN' DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN

*_#f*.**.**_# *_t **..**** .***.*** *_t._;******_:*****_#***_; .* *. YAB WSNVQIRNHLKNTATNLGNTTQFGSGLVNAEAATR

BPN' TNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ

Alignment of Subtilisin sendai to BPN': 55,6% identity. CLUSTAL W (1.7) multiple sequence alignment sendai NQVTPWGITRVQAPTAWTRGYTGTGVRVAVLDTGIS-THPDLNIRGGVSFVPGE-PSYQD

BPN' AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD

* _t * . *_;....**. _;.****_;>*.***_:*_;**_> .****., ** _# * _; ** _# * *_<;** sendai GNGHGTHVAGTIAALNNSIGWGVAPNAELYAV VLGANGSGSVSSIAQGLQWTAQNNIH

BPN' DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIE AIANNMD

_* *_*****_**._******_***._**** . _****_**** *** * * ._*..*. **_:. sendai VANLSLGSPVGSQTLELAVNQATNAGVLWAATGNNG SGTVSYPARYANALAVGAT

BPN" VINMSLGGPSGSAAL AAVD AVASGWWAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV

* * . *** _# * ** .*. **..*_< .**.****.**.* *_t**_a**_#.*_{a #}.****_> sendai DQNNNRASFSQYGTGLNIVAPGVGIQSTYPGNRYASLSGTSMATPHVAGVAALVKQKNPS

BPN' DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN

* _# . * .*****. * _# *...**** **** ***.*_{> ; ^}Λ**** *****^**. .*:*. sendai SNTQIRQHLTSTATSLGNSNQFGSGLVNAEAATR BPN' WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ

_*._***._{* * *}._{* **}._* ._{* **}._* ._**..

These alignements reveal that that homology between various sub- tilisin 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 IB) .

Sequence identities between different pairs of proteases are given below:

Sequence identity to BPN'

Savinase 60. .4%

Alcalase 69, .5%

BLAPR 60, .4%

ProteaseC 60. .4% ProteaseD 60. .0%

ProteaseE 58. .2%

Protease A 60, .0%

Properase 59. .6%

Relase 61, .5% PD498 44, .8% sendai 55, .6%

YAB 55 .3%

Sequence identity to Savinase: Alcalase 60.9% BLAPR 98..1%

ProteaseC 98, .5%

ProteaseD 98, .9%

ProteaseE 96, .7%

5 Protease A 97. .8%

Properase 98. .9%

Relase 98. .1%

PD498 44. .3% sendai 81. .4% o YAB 81. .8%

Structures

The protein structure of PD498 is disclosed in WO98/35026 (Novo 5 Nordisk) . The structure of Savinase can be found in BETZEL et al, J.MOL.BIOL., Vol. 223, p. 427, 1992 (lsvn.pdb) .

Homology modelling

0 Three dimensional structural models of the subtilisins prop- erase, 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 5 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 (Sali, A.; T.L. Blundell, "Definition of general topological equivalence in protein structures: A procedure involving comparison of properties and relationships through simu- 0 lated 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 Figure 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 ^xmodeller 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 "ltib" , available in Structural Classification of Proteins (SCOP) on the Internet..

Amylase :

The amylase used in the examples is the alpha-amylase of Bacillus halmapalus (W096/23873) , 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 W096/23874. 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 (W096/23873) . 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 a-amylase may be derived from a strain of Bacillus sp. DSM 12649. The strain was deposited on 25th January 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 Microorgan- ismen und Zellkulturen GmbH (DSMZ) , Mascheroder Weg lb, D-38124 Braunschweig DE.

Laccase :

The laccase used in this invention is that from Coprinus cinereus

(W098/38287) , 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

W098/38287.

Cellulase:

The cellulase sequence and structure used in the present invention is that of the core fragment of endoglucanase V from Humi- cola insolens (aka Cel45 or Carezyme) . The core fragment structure is available as 3eng.pdb (G.J.DAVIES et al . ACTA CRYSTAL- LOGR. ,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 W091/17243 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-length protein. „...,. ... . .:..-_. .. . IW/UR .14 JUIM UU!

¹²⁸

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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 pro- teins, were screened for their capacity to bind purified specific rabbit IgG, 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 im uno- beads (Dynal AS) loaded with pig anti-rabbit IgG, mouse anti-rat IgGl or IgE, or rat anti-mouse IgGl or IgE antibodies.

The respective phage libraries were incubated with the IgG, IgGl and IgE antibody coated beads. Phages, which express oligopeptides with affinity for rabbit IgG, or rat or mouse IgGl 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, TGI) were infected with M13-derived vector in the presence of a helper-phage (e.g. M13K07) , and incubated, typically in 2xYT containing glucose or IPTG, and appropriate antibiotics for selection. Finally, cells were removed by centrifugation. This cycle of events was re- peated 2-5 times on the respective cell supematants. After selection round 2, 3, 4, and 5, a fraction of the infected E.coli was incubated on selective 2xYT 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 con- sidered 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 o (mouse) , ra (rat) and hu (human) .

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SUBSTITUTE SHEET Table 2: Savinase antibody binding peptide sequences, epitope patterns and epitope sequences

YKVSAL 'Protein fragments a-amylase inhibitor jsavinase Y91 K27 V26 S24 G23J-21 sav14.0-pd14.0 TGKYVS Protein fragments Hu ^a-amylase jnhibitor_ isavinase 7S24 G25 K27 Y91 V93 iay17.0-pd17.1-17.2 Hu

D . O *- i

I 1

Table 4: Antibody binding peptide sequences, epitope patterns and epitope sequences for the T lanuglnosus lipase (Lipolase) .

LRSVYQ Protein fragments a-amylase jlipolase .L14 R81 S79 V77 Y16 Q15 •Iip13.0 ,Hu

/inhibitor ', j SGPWSW Protein fragments | .a-amylase^{" "} lipolase ^" isi70 G172 P174 W89 S83 Jip14.0 Hu inhibitor , '

ω a

03

H C H m GO m

2i

Table 5: Amylase (Natalase) antibody binding peptide sequences, epitope patterns and epitope se quences .

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

;CITSGPRAGNCG jPhage display G>>AG icarezyrπe icarezyme ;T95/S96 G27 P98 A100 G101 carl^ Ra ^■CITSGPRAGNCG iPhage display !> G > > A G •carezyme irarezyme P23 R201 A83 G84 car1.1 Ra

Table 7: Laccase {Mycelioptora ther opila laccase) antibody binding peptide sequences, epitope patterns and epitope sequences .

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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, ebLite 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 ac- ids, where each amino acid was situated within a distance of 5A 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 ac- ids situated within a distance of 5A 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 sur- face 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: L75 D41 Q2 179 and L42 D41 Q2 179. A shorthand notation for such a situation

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 epi- topes 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 vl (WO99/47680) was used as the parent protein for identification of epitope sequences that may cross react with enzyme epitopes. The structural coordinates from lBVl.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 vl : Bet vl 1.1: P31 A34 P35 A37 P59 S39/S40; Bet vl 1.2: P63 L62 P59 A37 P35 S39/S40; and Bet vl 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 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 epi- tope-antibody interaction, were defined around the identified epitopes. Practically, all amino acids situated within 5A 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 :

savl .1 Al Q2 S3 P5 H39 P40 D41 L42 N43 G63 T66 H67 A69 G70 T71 A73 A74 L75 N77

S78 179 G80 V81 L82 G83 N204 V205 Q206 S207

T208 Y209 P210 S212 T213 Y214 A215 S216 L217

savl.2 S153 G154 N155 S156 G157 A158 G160 S161 1162 S163 A169 R170 A174 M175 A176 V177 G178 R186 F189

S190 Q191 Y192 G193 A194 G195 L196 D197 1198 V199

T220 R247 K251 A254 T255 S256 T260 N261 L262 Y263

G264 S265 G266 L267 sav2.1 W6 G7 18 RIO Vll Q12 A13 P14 A15

A16 R19 L21 V84 T180 D181 Q182 N183 N184 1198

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 1162 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 241 S242

N243 V244 Q245 1246 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 Llll 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 135 S37 T38 H39 P40 D41 L 2 N43 144

R45 G46 T66 A69 G70 T71 172 A73 A74 L75

N76 N77 179 G80 V81 L82 G83 V84 A85 P86 S87 A88 E89 L90 Y91 A92 T208 Y209 P210 S21

T213 Y214 sav5.1 Al Q2 S3 V4 135 S37 H39 P40 D41

L42 N43 144 T66 A69 G70 A73 A74 L75 N76 N77 S78 179 G80 V81 L82 G83 P86 L90 T208

Y214

sav5.2 V30 T33 G34 135 S37 T38 L42 N43 144 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 18 S9 RIO Vll Q12 A13 P14 A15 A16 R19 N269 A270 E271 A272 A273 T274 R275

sav5.4 Al Q2 P40 D41 F50 L75 N77 S78 179 G80 V81 V104 S105 S106 1107 A108 Q109 GUO Llll 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 135 S37 T38

H39 P40 D41 L42 N 3 144 R45 G46 G47 Q59 T66 A69 G70 A73 A74 L75 N77 179 G80 V81

L82 A88 E89 L90 Y91 N117 G118 M119 H120 VI2

S207 T208 Y209 P210 G211 S212 T213 Y214 A215

sav7.1 K27 L31 1107 A108 Q109 GUO Llll E112 W113 A114 G115 N116 N117 G118 Mil9 A122 L124 L135 Q137

A138 V139 S141 A142 R145 V149

sav7.2 V104 1107 A108 Llll 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 Llll 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 1246 R247 L250

sav9.2 L126 G127 S128 P129 A152 S153 G154 S161 1162 S163 Y167 P168 A169 R170 Y171 A172 A176 V177 G178

Q191 Y192 G193 A194 G195 L196 D197 1198 V199 T260

N261 L262 Y263 G264

savlθ.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

savlθ.2 Vll 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

savlθ.3 L31 D32 H64 V68 V95 L96 1107 Llll 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 1246 R247 L250

savlθ.4 P131 S132 A133 L135 E136 V139 A151 A152 S153 G160 S161 1162 S163 Y167 P168 A169 R170 Y171 A172 N173 A174 A176 Q191 Y192 G193 A194 G195 L196 R247 S259 T260 N261 L262 Y263 G264

savll.O 6 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

savl2.0 L31 1107 A108 Q109 G110 Llll 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

savl3.0 Q2 S3 P5 T38 H39 P40 D41 L42 N43

H67 G70 A73 A74 L75 N77 179 G80 V81 L82 G83 V205 Q206 S207 T208 Y209 S212 T213 Y214 A215

S216 L217

savl4.0 A16 H17 R19 G20 L21 T22 G23 S24 G25

V26 K27 V28 A29 V30 135 144 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

savlδ.O 6 RIO 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

savlδ.O A13 A16 H17 G20 L21 T22 G23 S24 G25 V26 V28 172 A73 V84 A85 P86 S87 A88 E89

L90 H120 G229 A230 A231 A232 L233 V234 K235 Q236

K237 N238 P239 S240 W241 1246 H249 L250 A270 A273 T274 savl7.0 T22 G23 S24 G25 V26 K27 V28 A29 V30

L31 D32 135 144 R45 G46 G47 A48 F50 S87

A88 E89 L90 Y91 A92 V93 K94 V95 GUO 113

N117 G118 M119 H120 V121 A232 K235 Q236

savlβ.l W6 G7 18 S9 RIO Vll Q12 A179 T180

D181 Q182 N183 N184 N185 R186 A187 1198 V199 A200

P201 V203 H226 V227 A230 H249 L250 K251 N252 T253

A254 T255 S256 L257 S265 G266 L267 V268 N269 A270

savl8.2 A13 A16 H17 L21 T22 G23 V26 V28 V84

A85 A88 V121 L148 Y171 A172 N173 V174 M175 A176

G195 L196 D197 1198 V199 V227 A228 G229 A230 A231 A232 L233 V234 K235 Q236 K237 N238 241 N243 V244

Q245 1246 R247 N248 H249 L250 K251 N252 T253 A254

Y263 G264 S265 G266 V268 A270 A273 T274

savl9.1 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

savl9.2 Al Q2 S3 V4 P5 D41 H64 H67 G70 T71 A74 L75 N77 S78 179 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 : pdl.l D105 A108 S109 GllO llll R112 Y113 A114 A115 D116

Q117 N131 S132 T133 T134 L135 K136 S137 A138 V139

D140 Y141 A142 143 N144 K145 G146 A147

pdl.2 C128 E129 A153 G154 N155 D156 N157 V158 S160 R161

T162 F163 Q167 S170 G178 A179 1180 D181 D184 R185

K186 A187 S188 F189 S190 N191 Y192 G193 T194 W195

V196 T220 T262 N263

pdl.3 F50 L104 D105 S106 1107 A108 S109 GllO llll R112

Y113 A114 A115 D116 Q117 T133 T134 L135 K136 S137

A138 V139 D140 Y141 A142 W143 N144 K145 G146 A147

pdl.4 T28 *28aV A29 V30 D32 S33 G34 V35 Y37

*44aaV 145 K46 G47 Y48 D49 F50 151 R53

D54 N55 N56 P57 M58 D60 L61 K89 190 L91

A92 V93 R94 V95 L96 D97 A98 Y113 A114 Qll A119

P< 11.5 D32 S33 G34 K46 G47 Y48 D49 F50 151 D52

R53 D54 N55 P57 M58 L61 L91 A92 V93 R94

V95 L96 D97 A98 L104 D105 S106 1107 A108 S109

GllO llll R112 Y113 A114 A115 D116 Q117 G118 A119 T133 T134 L135 K136 S137 A138 V139 D140 Y141 A142

pd2.1 V19 T21 llll 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 1246 R247 Q248 A249 A273 V274 R275 Y276 pd2.2 S24 S25 T26 Q27 T28 *28aV L42 A43 R44 *44aK

*44aaV 145 D75 N77 D87 T88 K89 190 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 llll A115 G118 A119 K120 V121 L122 S137 A138

V139 D140 Y141 A142 W143 N144 K145 G146 A147 V148

V149 V150 1175 A231 A232 A235 S236 N243 1246 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 1246 S270 N271 K272 A273 V274

R275 Y276

pd3.1 L31 K46 G47 Y48 F50 L91 V93 S103 L104 D105

S106 1107 A108 S109 GllO llll 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 1246 R247 Q248 A249 1250 Q252 T253 K272

A273 V274 R275 Y276

pd4.1 W-6 S12 T13 P14 A15 A16 W17 D18 VI9 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 1246 R247

Q248 A249 1250 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 1246 R247 Q248 A249 1250 S270 A273 V274 R275 Y276

pd4.3 T26 Q27 T28 *28aV A29 V30 L31 Y37 *44aaV

145 K46 G47 Y48 D49 D52 R53 D54 N55 N56 P57 M58 V72 T88 K89 190 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 1107

A108 S109 GllO llll 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 145 K46 G47 Y48 F50 N55 N56 P57 M58 K89 190 L91 A92 V93 A108 S109

GllO llll R112 Y113 A114 A115 D116 Q117 G118 A119 L122 pd5.0 F50 S103 L104 D105 S106 1107 A108 S109 GllO llll

R112 Y113 A114 A115 D116 Q117 T133 T134 L135 K136

S137 A138 V139 D140 Y141 A142

pdδ.l Y4 Y6 G7 G63 H64 H67 V68 T71 N155 A179

F189 P201 G202 V203 N204 1205 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 190 L91 A119 V121 L122 N123 T208 A228 A231

pdδ.O W-6 T13 A16 W17 T21 R22 G23 Q27 *44aK A73

A74 *75aT G83 M84 A85 P86 D87 T88 K120 V121

1175 A176 V177 G178 V196 D197 V198 T199 A200 V227 G229 L230 A231 A232 L233 L234 A235 S236 Q237 G238

K239 N240 N243 Q245 1246 Q248 A249 1250 Q252 T253

A254 F264 Y265 G266 1268

pd9.0 W-6 Y6 G7 P8 Q9 N10 Til S12 T13 P14

A15 A16 W17 D18 V19 T21 M84 V139 W143 V148

V149 A151 P168 A169 Y171 P172 N173 A174 1175 A176

D181 S182 N183 D184 D197 P201 L230 L233 L234 K239

N240 N243 V244 Q245 1246 R247 Q248 A249 1250 E251 Q252 T253 A254 K267 1268 N269 S270 N271 K272 A273

V274 R275 Y276

pdlθ.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 1175 A176

N191 Y192 G193 T194 W195 V196 T262 N263 F264 *264aK

pdll.O W-6 S-5 Y2 Y4 Q5 Y6 G7 P8 Q9

N10 Til S12 T13 P14 W17 D18 V19 T21 A82

M84 1180 D181 S182 N183 D184 P201 G202 V203 N204

1205 H226 L233 S270 N271 V274 R275

pdl2.0 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 1175 A176 V177 G178 N191 Y192 G193 T194 W195 V196 D197 V198 T199 A200

V227 R247 1250 E251 A254 N263 F264^' *264aK Y265

G266 1268

pdl3, .1 W-6 S-5 P-4 D-2 P-l Yl Y2 S3 *3aA

Y4 Q5 P8 Q9 S12 T13 P14 A15 A16 W17

D18 VI9 T21 R22 G80 V81 A82 N271 V274 R275

pdl3.2 W-6 S-5 P-4 N-3 D-2 P-l Yl Y2 S3

*3aA Y4 Q5 P8 Q9 P14 W17 D41 G70 A74

D75 *75aT N76 N77 G78 179 G80 V81 A82 G83

A206 S207 T208 Y214 pdl4..0 T28 V35 D36 Y37 N38 H39 P40 D41 L42

A43 R44 *44aK *44aaV 145 K46 G47 Y48 D49

F50 R53 D54 N55 N56 P57 M58 T66 A69 G70

A73 A74 D75 K89 190 L91 A92 V 3 R94 Yll

T208

pdl5, .0 V30 L31 D32 S33 G34 V35 D36 Y37 N38

H39 L42 A43 *44aaV K46 Y48 D 9 F50 151

N56 P57 M58 D60 L61 N62 G63 H64 G65 T66

A69 190 A92 V93 R94 V95 L96 D97 A98 G100

SlOl G102 S103 S106 1107 GllO S125 L126 V209 P210 N211 N212

pdl6. .0 W-6 S-5 P-4 N-3 Y2 G7 P8 Q9 N10

Til S12 T13 P14 A15 A16 W17 D18 VI9 T21

R22 *75aT N76 A82 G83 M84 A85 P86 L233 N269

S270 N271

pdl7.1 Til S12 A15 A16 D18 V19 T21 R22 G23

S24 Q27 L230 A232 L233 L234 A235 S236 Q237 G238

K239 N240 N243 Q245 1246 Q248 A249 Q252 T253 N269

S270 N271 272 A273 V274 R275 Y276

pdl7.2 A108 llll 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 pdlβ.l W-6 T13 A16 W17 VI9 T21 R22 G23 S24

S25 *44aK M84 A85 P86 D87 T88 K89 G229 L230

A231 A232 L233 L234 A235 S236 Q237 K239 A249 1250

T253 N269 S270 N271 K272 A273 V274 R275 Y276

P >dl8.2 D-2 V30 V35 D36 Y37 N38 H39 P40 D41

L42 A43 R44 *44aK *44aaV 145 K46 G47 Y48

P57 T66 A69 G70 A73 A74 D75 *75aT N76 N77

179 V81 A82 A85 P86 D87 T88 K89 190 L91

A92 V93 R94 T208

For Lipolase, the following amino acid residues belong to the epitope area that correspond to each epitope sequence indicated in Table 4 :

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 1166 G190

lip2.2 V2 L6 F10 A173 P174 R175 A182 L193 Y194

R195 1196 T197 P204 R205 Y213 S214 H215 S216 S217

P218 E219 Y220 W221 1222 1235 V236 K237 1238 E239

1241 D242 A243 G246 N247 N248

lip2.3 V2 L6 F10 A182 L185 T186 L193 Y194 R195 1196 T197 H215 S216 S217 P218 E219 Y220 W221 1222 1235 V236 K237 1238 E239 G240 1241 A243 G246 N247 N248

lip2.4 V2 L6 F10 L193 Y194 R195 1196 T197 S216 S217 P218 E219 Y220 W221 1222 1235 V236 K237 1238 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 1202

P204 R205 L206 P207 P208 R209 E210 F211 G212 Y213

S214 H215 S216 S217 P218 E219 1238 E239 G240 1241 D242 A243 T244 G245 N248 ?R259? P250 N251 1252

P253 D254 1255

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 1241 D242 N248

lip5.1 A20 Y21 N25 N26 T50 F51 L52 Y53 S54

F55 E56 V63 T64 G65 F66 L67 A68 L69 176

V77 L78 S79 F80 R81 G82 S83 R84 S85 186

E87 N88 W89 K127 V128 A131 H145 S146 L147 G14

L151 G266

lip5.2 K94 F95 L96 L97 K98 E99 R108 G109 H110 Dill G112 R175 V176 G177 N178 R179 A180 F181 A182 E183 F184 R205 P207 P208 R209 E210 F211 G212 Y213 S214 H215 S216 1241 D242 N248

lip6.0 Q9 F10 Nil 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 1196 T197 D201 V203 P204

L206 P207 H215 H258 Y261 F262 1265

lip7.0 F13 A14 Q15 Y16 S17 A180 A19 A20 Y21

C22 G23 N25 N26 134 C36 A40 C41 F51 L52 Y53 S54 F55 E56 V63 T64 G65 F66 L67 S79

F80 R81 V120 A121 D122 T123 L124 R125 Q126 K12

V128 L264 1265

lipδ.l L12 F13 A14 Q15 Y16 S17 A18 A19 A20

134 V44 A49 T50 F51 L52 F66 L67 A68 L69

D70 N71 T72 N73 K7 L75 176 V77 S79 H135

P136 D137 Y138 R139 V140 V141 T143

lip8.2 L12 F13 A14 Q15 Y16 S17 A18 A19 A20

134 V44 A49 T50 F51 L52 Y53 S54 F55 G65

F66 L67 A68 L69 D70 N73 L75 176 VI 1 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 176 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 1166 D167

V168 F169 S170 G190 G191 T192 L193 Y194 R195 1196 Y220

liplθ.0 Nil L12 Q15 Y16 134 T35 C36 C41 P42 E43 V44 E45 K46 A47 D48 A49 D70 N71 T72

N73 K74

lipll.O F95 L96 L97 K98 E99 1100 N101 D102 C107

R108 G109 H110 Dill 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 1241 D242 A243 T244 N248

lipl2.0 L96 L97 K98 E99 1100 N101 D102 C104 S105 G106 C107 R108 G109 H110 T114 S115 V176 G177 N178 A180 F181 F184

lipl3 .0 Nil L12 F13 A14 Q15 Y16 S17 A182 A19

A20 Y21 N26 134 C36 A40 C41 P42 E43 V44

A49 F55 E56 V63 T64 G65 F66 L67 A68 D70

N73 L75 176 V77 L78 S79 F80 R81 G82 S83

R84 W89 W117 L124 V128 V141 F142 T143 G144 H14

S146 L147 G148 G149 A150 L151 A152 A155 lipl4.0 Q9 ^ F10 Nil F13 A14 S17 Y21 R81 G82

S83 R84 S85 186 E87 N88 W89 190 G91 N92

L93 F113 T143 G144 H145 S146 L147 G149 A150 T153 V168 F169 S170 Y171 A173 P174 R175 V176 L193 Y194

R195 1196 T197 D201 V203 P204 L206 P207 H215 H258

Y261 F262 1265 G266

lip15 .0 Nil L12 F13 A14 Q15 Y16 S17 A18 A19

A20 Y21 C22 G23 K24 N25 N26 D27 A28 134

T35 C36 A40 C41 P42 E43 V44 E45 K46 A47

A49 F51 L52 Y53 S54 F55 E56 T64 G65 F66

L67 S79 F80 R81 T123 L124 K127 L264 1265

liplδ.O A14 E87 190 H145 G172 1196 T197 H198 T199

N200 D201 1202 P204 R205 W221 1222 K223 S224 G225

T226 G246 N247 N254 1252 P253 D254 1255 P256 A257

H258 L259 W260 Y261 F262 G263 1265

lipl7.0 El 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 1222 K223

S224 G225 T226 V228 P229 V230 T231 R232 N233 D234 1235 V236 K237 1238 E239 G240 1241 D242 A243 T244

G245 G246 1262 liplδ.O Q9 F13 Y16 T32 N33 134 C41 P42 E43

V44 E45 K46 A47 D48 A49 T50 F51 L52 L67

A68 L69 D70 N71 T72 N73 L75 176 V128 V132 H135 P136 D137 Y138 R139 V140 V141 F142 Y164 D165

1166 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 :

jel.l N2 G3 T4 R33 P346 Y349 1352 L353 T354 R355

P360 V362 D366 Y367 M378 K379 A380 K381 1382 D383

P384 1385 L386 E387 A388 R389 Q390 N391 F392 A393

Y394 1450 T451

jel.2 Y57 D58 Y60 D61 F65 N66 Q67 L104 G105 G106

A107 D108 A109 T110 Elll 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 Wll 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

1370 P371 T372 H373 S374 V375 P376 A377 M378 K379

1382 D383 L386

je2.2 L289 L293 V314 P318 T323 F324 V325 D326 F339 K345

P346 L347 A348 Y349 A350 L351 1352 L353 T354 R355

F356 Q357 G358 Y359 P360 S361 V362 F363 Y364 G365

D366 Y367 Y368 G369 P376 A377 M378 K379 1382 1385 R389 Q397

je2.3 N102 V116 E117 V118 P120 R123 D159 G160 V161 D162

W163 Q168 F169 Q170 N171 R172 1173 Y174 K175 A182

W183 D184 V187 D188 N193 Y194 D195 Y196 L197 M198 Y199 A200 D201 V202 H236

je2.4 TI N2 T4 M6 Y8 D26 L30 R31 N32 R33

G34 135 V325 D326 F339 K345 Y349 L353 V362 F363

Y364 G365 D366 Y367 Y368 G369 1370 P376 A377 M378 K379 1382 D383 P384 1385 L386 E387 A388 R389 Q390 N391 F392 Y394 H417

je3.1 M6 Q7 Y8 F9 E10 L13 H19 W20 N21 R22 L23 R2 D25 D26 A27 S28 N29 L30 R31 N32

R33 1385 W39 140 P41 P42 A43 W44 V52 G53

Y54 Y75 A87 L88 N91 V93 D98 V100 Y364 Y36

e3.2 Y8 F9 Wll H19 W20 W39 140 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 1231 D232 A233 V234 K235 H236 1237 E262 H328

je3.3 Y8 F9 HI9 W20 W39 140 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 TllO Elll A135 W136 T137 K138 F139 D140

F141 P142

je4.1 L23 D25 D26 A27 S28 N29 L30 R31 N32 R33

G34 135 T36 138 A84 185 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 (e.g. "SDFGHKV") 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 18A, preferably 15A, more preferably 12A, is then claimed to be an epitope. The relevant distance can easily be measured using e.g. molecular graphics programs like In- sightll 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 pro- tein 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 Mo- lecular Principles, W.H. Freeman and Company, NY, ISBN: 0-7167- 1566-X (1984) .

Substitutions of one or more residue (s) within 18A, prefereably 15A, more prefereably 12A, 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, NY) .

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 :

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 μl 0.9% (wt/vol) NaCI (control group), or 50 μl 0.9% (wt/vol) NaCI containing 10 μg of protein. Blood samples (100 μl) were collected from the eye one week after every second immunization. Serum was obtained by blood clothing, and centrifugation.

Specific IgGl and IgE levels were determined using the ELISA specific for mouse or rat IgGl 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 IgGl and/or IgE responses in mice.

Epitope: A172/A169 R170 A194 G193 N261 Pattern: A R > R > A > N Antibody: IgGl + IgE Backbone : Savinase

The variant carried mutation R170F.

In a competitive IgE ELISA, this variant was less effective in competing for anti-savinase antibodies, giving a 15% lower end- point 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 IgGl levels were not significantly affected.

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

The variant carried 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 IgGl 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 epi- tope 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: IgGl Backbone : Savinase

The variant carried 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 IgGl 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 anti- bodies (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 immuno- genicity 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 IgGl and IgE antibody levels in mice, and an inhibiting effect on IgG binding, re- spectively.

Epitope area: P131, S132, A133, L135, E136, V139, A151, A152, S153, G161, S162, 1165, 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 Y167I.

In a competitive IgE ELISA, the variant was less effective in competing for anti-savinase antibodies, giving a 8% lower end- point 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 IgGl levels were dramatically increased (p<0.01).

Epitope: T143 N173 N140 E136 L135 Pattern: S/T N N > E L Antibody: IgGl Backbone : Savinase Epitope area: V10A, 1107, A108, Llll, 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 Bo- rate 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, 5mM Succinic Acid, 150 mM NaCI, 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 proce- dures. 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 com- petitior) .

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

Epitope: T143 N173 N140 Ξ136 L135 Pattern: S/T N N > E L

Antibody: IgGl

Backbone : Savinase

Mutation: E136K Modification: bis-NHS-PEGlOOO

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 vl; Der f2; Der p2 and Phi p2) , based on their structures (lbtv.pdb; lahm.pdb; al9v.pdb; and lwhp.pdb, respectively) , sequences (SEQ ID NO : 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 vl from Betula pendula) , House dust mites (Der f2 from Dermato- phagoides farinae and Der p2 from Dermatophagoides pteronyssi- nus) , and Timothy grass (Phi 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 betweenthe alfa-carbon atoms of subsequent amino acids, ii) a minimal accessability of the amino acid of

20A2, iii) the largest maximal distance between the most distinct amino acids should be less than 25A 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 vl:

Epi#02

A146, K32, Q36, F30, T142, R145, V12

A34, K32, Q36, F30, T142, R145, V12

Epi#03

L62, K65, , 156, Y66

L24, K20, H76, 123, Y81

L24, K20, H76, 1104, Y81

Epi#04

K134, S136, Q132, K129, A130, A135

K134, S136, Q132, K129, V128, Gl

Epi#05

G140, A146, R145, T10, Gill, A106, T107, V12 G26, A146, R145, T10, GllO, A106, T107, V12 G140, A146, R145, T10, GllO, Sll, S149, L152 GllO, A106, Sll, T9, G140, R145, T10, V12 G140, A146, R145, T10, Gill, Sll, S149, V12

Epi#06

GllO, P108, D109, T107, A106, P14

Gill, 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, Gl, V2 , H121, F3

K65, E60, F64, V67, F58

K65, E60, F58, V67, F64 K129, E127, Gl , 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, TIO, R145, T142, F30, G26, K32

Epi#ll

F30, K32, 138, Q36, V33, E148 F22, F30, 138, Q36, V33, E148 F30, L143, 138, 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, GllO, Gill A16, R17, P14, T107, A106, P108, GllO, Gill A157, R17, P14, T107, A106, P108, Gill, GllO

Epi#15

K65, P90, D93, 191, K97, G92

K32, P31, D27, 156, K65, G61

Epi#17

A153, S149, R145, Sll A106, Sll, 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 5 D125, K123, E96, P90, F64, P63, K65

Epi#25

R70, K55, 144, E45, E42

R70, K54, 144, E45, N47 io R70, K68, 153, E45, N47

Epi#27

D93, E127, D125, K123

15 Epi#28

A146, Q36, F58, E60, L62, F64, P63, K65 138, Q36, F58, E60, L62 , F64, P63 , K65 A34, Q36, F58, E60, L62, F64, P63, K65 L143, Q36, F58, E60, L62 , F64, P63 , K65

20 V33, Q36, F58, E60, L62 , F64, G61, K65

Epi#29

G61, K65, L62, F58, E60

156, K65, L62, F64, E60

25 G89, K65, L62, F64, E60

V67, K65, L62, F64 , E60

Ξpi#30

Gl, N4, S99, H121, K97, 191, P90 30 1113, 113, S149, H154, S155, L152, L151 113, L152, A153, H154, S155, L151, V33 GllO, 113, S149, H154, S155, L152, L151 Gl, N4, S99, H121, K97, 198, V2 Gl, N4, S99, H121, K97, 191, V85 Epi#33

K32, F30, P35, S39, S57, K65

Q36, F30, P35, S39, S40, K32

5 K32, F30, P35, S40, S57, K65

K65, F58, P35, S39, A34, R145

Epi#34

V105, P14, T107, V12, R145, Y150, S155 10 1113, P14, T107, V12 , R145, Y150, S155

Epi#37

P50, V74, L24, R17, N159

P50, V74, L24, K20, N159

15 P14, R17, L24, K20, N159

Epi#38

L143, G140, E141, R145, V33, N28, P31, S39

L143, G140, E141, R145, V33, N28, P31, S40

20 L143, G140, E141, R145, V33, N28, P31, S57

Epi#39

A130, E127, H126, T94, P90, G89, L62

A130, E127, H121, T94, P90, G89, L62

25

Epi#40

A157, L152, A153, Y150, K32, S39 A153, L152, A157, Y150, K32, S40 R17, L151, A153, Y150, K32, S40 30 R145, L143, A34, Y150, A153, S155 R145, L143, G140, T9, K115, TIO

Epi#41

P63, Y66, L62, S57 Epi#44

123, R17, D156, Y150, S149, V12, TIO

L24, R17, D156, Y150, S149, V12, P14 L24, R17, D156, Y158, A16, A106, P108

113, R17, D156, Y150, S149, V12, TIO L151, R17, D156, Y150, S149, V12 , TIO

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, SlOl, 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, 113, Y86

L40, K14, A39, 188, 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, SlOl, R128, A98, P99, D129 T107, S57, D59, SlOl, 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, 168, H74, F75, T70, N71 N114, N46, D113, K48, N71, T70, T49

G83, N46, D113, K48, N71, T70, T49

Epi#10

L40, 113, D42, N44, V81, K48, N46, N114, G115 L40, 113, D42, N44, V81, K82, N46, N114, G115 L37, D19, G20, V18, V3 , D4 , K6 , A120, T107, V105

Epi#ll F75, K51, llll, Q45, V116, D113 F75, K51, llll, Q45, V81, D113

Epi#12 Y90, E38

Epi#13

H30, R31, P95, A98, P99, SlOl, G60, L61

Epi#15 K96, P99, D129, 128, R128, A98

K96, P99, D129, 1127, R128, A98

K96, P99, D129, 129, R128, A98

K55, P66, D64, 168, T70, G67

Epi#18

R31, R128, 128, G125, T123, H124, V105 R31, R128, 1127, G125, T123, H124, V105

Epi#22 DI, M17, D4, V3, K6

DI, M17, D19, P34, K96

DI, M17, D4, V5, K6

Epi#23 K14, Nil, E12, N44, Q85, P79

K14, Nil, E12, N10, Q45, P79

K14, Nil, E12, N44, Q84, P79

K14, Nil, 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 5 D129, K126, E102, P99, R31, P95, K96

Epi#25

R31, K96, 197, D59, E62 R128, R31, 197, D59, E102 io R128, K126, 1127, E102, N103

Epi#27

D64, E62, D59, K100

D59, E62, D64, K55

15 D87, E38, D19, K33

D19, E38, D87, K15

D19, E38, D87, K14

D19, E38, D87, K77

20 Epi#28

V16, D87, Q85, K14, E12, K15, Q2 , DI

113, D87, Q85, K14, E12, K15, Q2 , DI

V3, DI, Q2, K15, E12, K14, Q85, D87

L40, D87, Q85, K14 , E12, K15, Q2 , DI 25 188, D87, Q85, K14, E12, K15, Q2 , DI

V76, D87, Q85, K14, E12, K15, Q2,D1

V18, DI, Q2, K15, E12 , K14 , Q85, D87

Epi#29 30 G32, N93, L61, E62 V94, N93, L61, E62

Epi#30

G60, 197, A98, H30, K96, P34, P95 168 , N71 , H74 , K77 , P79 , V81 G32 , 197 , A98 , H30 , K96 , P95 , P34

Epi#34 V105, P26, S24, G125, R128, SlOl, P99 W92, P34, T91, V94 , R31, SlOl, P99 128, P26, T123, G125, R128, SlOl, P99

Epi#37 A120, V16, L40, K14 , Nil A39, V16, L40, K14, Nil Y90, A39, L40, K14, Nil Y86, A39, L40, K14, Nil

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, SlOl, 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, 113, Y86

L17, K89, A72, 188, Y90 L17, K89, A72, 152, Y90

Epi#04

K15, SI, Q2, K14, V16, L17 K15, SI, Q2, K14, A39, L17

K15, SI, Q2, K14, V40, 113

Epi#05

G60, A56, L61, P99, G32, R31, H30, 197 G60, A56, L61, P99, G32, R31, H30, 128

Epi#06

G60, A56, D64, S57, K55, P66 G83, N46, D114, T49, K48, P79 G60, N103, D59, SlOl, R31, P95

Epi#08

K55, D64, S57, V106, F35 K55, E62, S57, V106, F35

Epi#09

L61, G60, E102, R128, 128, K126, N103, T123, V105 L61, G60, E102, R128, 1127, K100, N103, T123, V105 L61, G60, E102, R128, 1127, 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#ll

K14, 113, Q85, V81, E42

K15, 113, Q85, V81, E42

K14, 113, 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, 168, K89, A72

K55, P66, D69, 168, K89, A39

K55, P66, D64, 154, K109, G115 K55, P66, D64, 154, K109, A9

Epi#18

R31, 129, A125, SlOl, E102, N103

R31, 129, A125, SlOl, E102, V104 R31, 129, 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, 128, E102, N103 R128, K126, 1127, E102, N103 R128, K126, 128, E102, V105

5 Epi#27

D64, E53, D69, K89

D69, E53, D64, K55

D59, E62, D64, K55

io 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

15 Epi#29

197, K100, L61, E62 G60, N103, L61, E62 1127, N103, L61, E62

20 Epi#30

G60, N103, SlOl, H30, K96, 197, P95

G60, N103, A125, H30, K96, 197, P95

128, 1127, A125, H30, K96, 197, P95

25 Epi#33

Q36, F35, V106, S57, A56, K55 K33, F35, V106, S57, A56, K55

Epi#34

30 128, P26, S24, G23, G20, T123, S57

128, 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, SlOl

Epi#41

P19, L17, V3, SI 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

1127, SlOl, R31, 197, A98, L61, N103, P99, P95

128, SlOl, R31, 197, 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, 128, R31, H30 T123, H124, K126, R128, 128, R31, H30 D4, H124, K126, R128, 128, R31, H30

Phi p2 :

Epi#02

T87, K85, Q61, S38, R34, R67

T87, K85, Q61, P63, R34, V42

Epi#03

K10, A90, 188, Y86

K10, A18, 188, Y86 Epi#04

R34, S38, Q61, K85, T87, 188

R34, S38, Q61, K85, T87, A90

Epi#05

G47, A18, S12, T87, G89, T91, T5 , VI

G73, A29, L69, T27, G50, T53 , T45, V42

Gil, A18, L20, T91, G89, A90, T87, 188

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, Gil, 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 , Gil

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 , VI

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

188, 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

188, N13, S12, H16, K15, P59, L60

188, N13, S56, H16, K15, L60, P59

188, N13, A18, H16, K15, P59, L60

Epi#33

K46, F54, V42, S56, K15

H16, F54, V42, S56, K15

Epi#34 VI, P2, T5, V4, P94, Y92 , T87 VI, P2, T5, L20, G89, T91, T87

V81, P94, T5, VI, 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, VI, P2

L69, R67, D79, P94, Y92 , A93 , VI, 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 , VI

E84, D80, P94, P2 , VI Epi#50

D39, W41, A43, T45

D39, W41, V42, T45

Epi#51

D79, H36, E84, T87, K10, Gil, H16

D39, H36, Q61, K85, P63, R34, W41

D79, H36, E40, D39, G37, R34, W41

Q6-1, H36, E84, T87, KIO, Gil, H16

Table 8: Each row indicates an epitope pattern. At each position (from 1 up to a maximum of 12) the cells indicate which amino acids (single letter coding) that-ar at that position. The last column indicates the patterns that were identified using IgE antibody binding.

r O C3)

Example 10

For this example the third-generation epitope sequences were determined in further 11 environmental allergens (Bosd2, Equcl, Gald -mutant (with alanine substituted for glycine in position 102), Hevb8, Profillinl-AC, Profillinl-AT, Profillin2-AC, Pro- fillin-birch pollen, Rag weed pollen5 and Vesv5) , based on their structures sequences (SEQ ID NO: 12, 13, 15, 16, 17, 18, 19, 20, 21 and 22, respectively), their structures (lbj7.pdb, lew3.pdb, lflu.pdb, lgδu.pdb, lprq.pdb, laOk.pdb, lf2k.pdb, lcqa.pdb, Ibbg.pdb, and lqnx.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 vl, Der f2, Der p2 , and Phi 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 (Profilinl-AC, a profilin isoform IA and Profilin2-AC, a profilin isoform II) , Arabidosis thaliana (Profillinl-AT a cytoskeleton profilin) , Birch (Profilin-birch pollen (Birch pollen profilin) , Rag weed pollenδ (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 alfa-carbon atoms of subsequent amino acids, ii) a minimal accessability of the amino acid of 20A2, iii) the largest maximal distance between the most distinct amino acids should be less than 25A iv) the best epitope were taken, v) the homology with the epitope pattern of in- terest was 100%

a. Epitopes that contain the same protein surface residue more than once. These are artefacts generated by the described algo- rithm. 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, 17, Y76

L134, K130, H92, 17, Y76

L128, K130, H92, 191, Y76

Epi#04 R72, Y76, S50, Q73, K71, V69, 145

K71, Y76, S50, Q73, R72, V69, L80

K71, Y76, S50, Q73, R72, V69, 142

Epi#06 G14, P13, D47, SIO, Kll, P9

G14, P13, D47, SIO, S94, P9

G14, P13, D47, C44, SIO, P9

Epi#08 K71, E49, S50, V69, F82 K71, E49, S50, V79, F82

Epi#09

17, SIO, D8, E95, K119, N96, S122, T121 SIO, 17, D8, E95, Kll, 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, T151, N153, C154, F55, R41, R17

Epi#ll

K26, 1145, Q132, E143 K26, 1145, Q132, E137

K26, 1145, Q132, E129

Epi#12 Y105, E108 Y83, E81

Epi#15

N153, P156, D152, 1149, T121, G120 R17, P156, D152, 1149, T121, G120 N153, P156, D152, 1149, R17, G14 Epi#18

R109, 1110, G107, Y83, T85, E81, V69

R109, 1110, G107, Y105, T85, E81, V69

5

Epi#19

E43, N46, S50, Q73, R72, K71

D47, N46, S50, Q73, R72, G75

E49, N46, S50, Q73, R72, K71

10 145, N46, S50, Q73 , R72, Kll

Epi#20

V30, K28, P34, L57, L65, K58, D59, G32, D27 V30, K28, P34, L57, L64, K58, D59, G33, D27 15

Epi#22

D8, SIO, D47, P13, E15

D8, SIO, D47, P13, E43

D47, SIO, D8, V93, E95

20 D8, SIO, D47, C48, K71

Epi#23

K119, N96, E127, S122, L128, P125 K150, N147, E146, Y20, F123, P125 25 Kll, N96, E127, S122, L128, P125

Epi#24

E129, K130, E126, P12Ξ, S122, L128, Q133

E126, K130, E129, P125, S122, R17, K119

30 E126, K130, E129, P125, T124, L128, Q133

Epi#25

R72, K71, 145, D47, N46

R72, K71, 145, E43, N46

35

Epi#27

D47, E49, E74, K71 D24, E143, E146, K150 D47, E43, E15, K119

40

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

45 G61, D59, Q60, K58, E31, K28, G32, D27

Epi#29

V69, K71, L80, R72, E74

145, K71, L80, R72, E74

50 G61, Q60, L64, F55, E68 C5 o o

3 a

H U α.

CM CM

IΛ IΛ

90 o en

#

-H ft r pq P

Epi#47

L128, E126, E129, L134, R138, Q133, N142, P141, S136

V69, E81, E68, 142, R41, F55, N37, R40, P156 V69, E43, E15, 142, R41, F55, N37, R40, P156

S122, E127, E129, L134, R138, Q133, N142, P141, S136

Epi#48

E43, D47, P13, P9, V93 SIO, D47, P9, P13, G14

E43, D47, P13, P9, V90

E49, D47, P13, P9, V93

Equcl:

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, 163, Y72

Epi#05

G35, A65, S69, T93, G97, R26, S112, Y123 G35, A65, S69, T93, G97, R26, S112, 125

Epi#07

G97, T93, S70, D91, S100, R110, V125, P132, D12< 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#ll

F133, K47, 1167, Q158, V163, E165

Epi#12 Y38, E142 Y38, E36 Y139, E142

Epi#13 5 K129, P132, D45, 1167, Q158, G161 R131, P132, D45, 1167, K164, G161

Epi#16

P87, Y72, R64, S70, S69, D67, A65, N68 10

Epi#17

A65, S31, R64, S34

Epi#18 15 R64, S31, 130, A65, S34, L66, N68, S69

Epi#19

E82, N80, C83, Q178, R175, K172

20 Epi#22

D130, P132, D128, Y106, K129

Epi#23

D144, K150, E148, P147, S146, E151, K155

25

Epi#25

R160, K159, 1156, E151, E148

Epi#27 30 E118, E142, D144, K172 E36, E142, D144, K172

Epi#28

1173, D174, Q178, L179, E85, C83, F177, G181, R180 35 1173, D174, Q178, L179, E85, C83, F177, P143, D144

Epi#29

G181, Q178, L179, R180, E36

G181, Q178, L179, R180, E85

40

Epi#30

130, N27, S112, H119, 1121, 125, V23

Epi#31 45 L122, R110, N27, R26, F28, 130, D96 L124, R110, N27, R26, F28, 130, D96

Epi#33

H119, Y38, V62, S34, S31, R64

50 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, Ξ118, 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

Kl, D87, S86, V2, F38

Kl, D87, S86, V2, F3 Epi#09

Epi#10

E7, All, R14, A10, C6, F3 , R5 , R125 D87, All, R14, A10, C6, F3 , R5 , R125 T47, N46, N44, S36, F34, R114, R112 D18, A10, R14, All, C6, F3 , R5 , R125 T118, N113, R112, A110, F34, R114, K116

Epi#ll L129, 1124, Q121, V120, D119

Epi#12 Y53, E35 Epi#15

R73, P70, D66, 178, A82 R73, P70, D66, 178, 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, 1124, E7, C6, F3 , All, R14 A122, D119, Q121, 1124, E7, C6, F3 , All, R14

Epi#29

G126, R125, L129, R128, E7

G16, R14, L129, R128, E7 Epi#30

1124, L129, A10, H15, 188, L84 1124, L129, All, H15, 188, L84

Epi#31 L75, R73, N65, R61, W62, 198, D101 L75, R73, N74, R61, W62, 198, D101

Epi#33

Q41, F38, V2, S86, S85, Kl Q41, F38, V2, S36, AllO, R114

Epi#34

W63, W62, T69, G71, R73, S72, P70

W62, W63, S72, L75, R73, T69, P70

Epi#36

AllO, A107, A102, S100, K96, A90, A82

Epi#37 A10, R128, L129, R14, D18 A10, R128, L129, K13, N19

Epi#40

R128, L129, All, T89, A90, S85 R14, L129, All, 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, AllO, R112, R114, Will, N27, Q121

F3, All, E7, R5, W123, D119, Q121

W123, A122, T118, R114, Will, N27, Q121

He b8 :

Epi#01

L20, P109, P112, K86, R84, N116, Y125, Q129, Till LllO, P109, P112, K86, R84, N116, Y125, Q129, Till

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, 175, Y72 Epi#05

G30, A48, L60, P62, G58, T63, H66, G69

G58, A61, R84, P112, G113, Till, S89, G88

G80, A81, F54, P79, G58, T63 , H66, G69

Gil , 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#ll

F54, L60, 183, 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, 1105, 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, 1105, D107, E108 R96, H28, 126, 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

1105, P112, S89, LllO, R19, T21, S37

1105, P112, Till, 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, LllO, G113, Till, P109, S89

R19, LllO, G113, Till, P112, S89

Epi#41

P62, L65, V47, S44 P109, Y106, LllO, S89 P112, Y106, LllO, S8 Epi#44

L20, R19, D16, W3 , Y6 , S2 , G17, P109

LllO, R19, D16, W3 , Y6, S2 , G17, P109

5 Epi#45

K52, P57, F54, R96, D124, L127 D55, P79, F54, R96, D124, L131

Epi#47 10 175, G77, E78, V82, R84, N116, P112, S89 175, G77, E78, 183, R84, N116, P112, P109

Epi#48

E78, Q76, P79, P57, G58

15 E78, Q76, P79, P57, G80

E78, Q76, P79, P57, G77

Epi#50

D9, W3, W33, S2, T5 20 D16, W3, W33, S2 , T5

Epi#51

R19, H18, E108, S89, K87, K71, H66

R19, H18, E108, D107, K87, K71, H66

25

Profillinl-AC:

Epi#01 30 L116, Nlll, P106, K80, K81, NlOl, S83, Q105, T108 L116, Nlll, P106, K80, K81, NlOl, YlOO, Q105, S83

Epi#02

T44, N51, P54, R56, T69, Y78, R71, V68 5 L24, K93, S92, R75, S76, Y78, R71, R56

Epi#03

L24, K93, 1121, Y119

L24, K90, 1121, Y119

40

Epi#04

K80, YlOO, S83, Q105, K103, NlOl, G82

K80, YlOO, S83, Q105, K103, T17, G12

K80, YlOO, S83, Q105, K103, T17, G14 5

Epi#05

G34, A33, A36, T38, G64, A63, H66, V68

G34, A33, S32, T17, G12, T4 , SI, Y5

50 Epi#06 A46, N50, 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#ll

F125, K93, 1121, Q123, D118 F125, K90, 1121, Q123, D118 F49, K90, 1121, Q123, D118

Epi#12 Y119, E114 YlOO, 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, 155, R56, A57

R56, P54, D53, 155, T69, A57

R56, P54, D53, 155, T44, A40 Epi#16

Q105, P106, YlOO, G14 , Q18, S32, A36, A33, D7 Q105, P106, YlOO, G14, Q18, S32, A36, A63, D61

Epi#17 AllO, 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

5

Epi#23

K103, NlOl, E102, S83, Q105, P106

K103, NlOl, E102, S83, Q105, A84 io Epi#24

E114, K115, AllO, P106, S83, E102, K103 D53, G59, A57, P54, R56, L70, K80 E102, K103, A15, P106, S83, A84, Q105

15 Epi#25

R71, R56, 155, D53, N50 R71, R56, 155, D53, N51

Epi#28 20 1104, Q105, K103, E102, K81, S83, K80 G107, Q105, K103, E102, K81, G82, K80 A84, Q105, K103, E102, K81, S83, K80 AllO, Q105, K103, E102, K81, S83, K80

25 Epi#29

1121, K115, L116, E114 V112, K115, L116, E114

Epi#30 30 G59, 155, S58, H66, K80, L70, V68 G59, 155, S58, H66, K80, P106, V99

Epi#33

K80, Y78, V68, S58, A57, R56 35 K81, Y67, V68, S58, A57, R56

Epi#34

155, P54, S58, V68, R71, Y78, P106

W29, W2, T4, Vll, G12, Y5 , SI

40

Epi#36

A63, A36, A33, Vll, G14 , YlOO, S83, Q105, K103, P106, AllO, A15

A63, A36, A33, Vll, G14, YlOO, T108, Q105, K103, P106, A15, AllO

45 Epi#37

A57, R56, L70, R71, Y78

A57, V68, L70, R56, D53

Y78, R71, L70, R56, N51

P54, R56, L70, R71, D73

50 T69, R71, L70, R56, D53 Epi#38

G82, E102, A84, V99, NlOl, 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, AllO, G107, T108

V68, R71, D73, Y78, AllO, G107, T108 L70, R71, D73, Y78, AllO, V112, T108

L70, R71, D73, Y78, AllO, 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, SI, T4

D7, Y5, W2, W29, SI

Epi#51

K80, H66, D61, T44, P39, T28, W29

K80, H66, D61, T38, P39, T28, W29 Profillinl-AT:

Epi#01

P109, P89, K86, R84, N116, Y106, Q114, Till

Epi#02

L42, K43, Q45, F66, T63, Y72, R84, V74

L42, K43, Q45, F66, T63, Y72, R84, V82 Epi#03

K96, 1127, Y125 K86, 175, 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, G5i V74, F66 K51, E55, G5c A61, F59

Epi#09

D29, D48, K52, F59, A61, T63 D29, D48, K51, F59, A61, T63

Epi#10

E108, Till, N18, T21, F39, G68, K71

E108, Till, N18, T21, F105, G112, K86

Epi#ll

F105, K86, 175, Q76, V82, E78 F66, K43, 147, Q28, V32, D29 F59, K52, 147, Q28, V32, D29

Epi#12 Y125, E130 Y125, E128 Epi#15

K43, P44, D29, 147, K52, G58

K43, P44, D48, 147, Q45, G49

K43, P44, D29, 147, 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, 183, D53, E55

R121, K95, 183, E78, V82

Epi#26

W33, S2, W3, V32, G30, D29

Epi#27 E128, E130, D124, K96 E130, E128, D124, K95

Epi#28

175, Q76, E78, Q79, P57, K51 A61, Q76, E78, Q79, P57, K52

V32, D29, Q99, E130, 1127, S129, D124

V32, D29, Q99, 1127, 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, MHO, H19, Q114, L117, V15 G113, MHO, H19, Q114, L117, V15

Epi#33 Q41, F39, P40, S36, A37, K38

Epi#34

V74, P62, M73, G88, P89, Y106, Till Epi#37

T111, V15, L117, R121, Y125 Till, V15, L117, R121, D124

Epi#39 A81, E55, P57, G58, L60 A81, E78, P57, G58, L60

Epi#40

R121, L117, G112, Y106, P109, Till R121, L117, G112, Y106, P89, Till

Epi#41

Y125, L131, S129 Epi#44

175, R84, Y72, A61, G58, P62 175, 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, Till, L117, R121, H10

D107, H19, E16, Q114, L117, R121, HIO D14, H19, D107, T21, K38, Q35, W33

Profillin2-AC: Epi#01

L116, Nlll, P106, K80, K81, NlOl, S83, Q105, T108 L116, Nlll, P106, K80, K81, NlOl, 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, 1121, Y119 Epi#04

K81, YlOO, S83, Q105, K103, Til , G12

K80, YlOO, S83, Q105, K103, A84, G82 K81, YlOO, S83, Q105, K103, T17, G14

K80, YlOO, S83, Q105, K103, NlOl, 1104

K81, YlOO, 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#ll

F125, K93, 1121, Q105, E102 F125, K93, 1121, Q123, D118

Epi#12 YlOO, 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, 1121, K93, G124

K103, P106, D118, 1121, Q105, G107 K103, P106, D118, 1121, 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, NlOl

R66, N58, R56, S57, V68, G82, S83, P106, NlOl 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, NlOl, E102, S83, Q105, P106

K103, NlOl, 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, NlOl

Epi#28

1121, 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

1121, K115, L116, E114

V112, K115, L116, E114

Epi#30

155, N47, A44, H24, K93, 1121, L116

155, N47, A43, H24, K93 , 1121, L116 Epi#31

R56, N58, R66, F60, V68, 155, D51 R66, N58, R56, F60, V68, 155, 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, Vll, G12, T4 , SI W29, W2, T4, G12, G14, T13, T8

Epi#37 T108, V112, L116, K115, Y119 T108, AllO, L116, K115, Nlll T13, V112, L116, K115, D118 P106, AllO, L116, K115, Nlll Epi#38 G64, E61, A40, V37, N27, P39, S38 G82, E102, A84, V99, NlOl, P106, S83

Epi#39 AllO, 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

SI, Q3, D7, W2, Y5, S32, G12 , T8 SI, Q3, D7, W2, Y5, A30, A36, P39

SI, Q3, D7, W2, Y5, S32, Vll, T8

SI, Q3, D7, W2, Y5, S32, G12, T4

SI, Q3, D7, W2, Y5, A30, A33, T31

SI, Q3, D7, W2, Y5, A30, A36, T28 SI, Q3, D7, W2, Y5, S32, G12, T13

SI, 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, SI 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, PH4, 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, 1129, Y127 Epi#04

S40, Q43, K45, T50, G32

S40, Q43, K45, T50, G51

S40, Q43, K45, T50, 149 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#ll

F56, K98, 185, Q78, V84, E122

F56, K98, 127, Q37, V34, D31

F56, K97, 185, 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, 149, Q47, G32

K45, P46, D31, 149, K54, G60

K45, P46, D31, 149, K54, G82

K45, P46, D31, 149, 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, LH, A23

R123, N118, E122, L124, LH, A36

R123, N118, E122, L124, LH, A24

10

Epi#24

E109, G90, EHO, P114, R86, E80, Q78 E57, K54, E58, P59, F56, A81, Q78 E58, G60, E57, P59, F56, E80, Q78

15

Epi#25

R86, K88, 1107, E109, EHO

R86, K88, 177, E80, V84

R86, K88, 1107, E109, V112

20

Epi#27

57, E58, D55, K54

D55, E57, E58, K54

25 Epi#28

V34, D31, QlOl, K98, E122, L128, Q131, G132, D130 1129, D126, Q131, L128, E122, K98, QlOl, GlOO, D130 172, H68, Q47, F44, E48, K45, Q43, G70, K73 172, H68, Q47, 149, E48, K45, Q43, G71, K73

30

Epi#29

1129, QlOl, L128, R123, E122

G132, Q131, L128, R123, E122

35 Epi#30

177, M75, A63, H61, P59, L62, P64 G90, M75, A63, H61, K54, L62, P64

Epi#33 40 Q116, Y108, PHI, S91, K89 K88, Y108, PHI, S91, K8

Epi#34

V76, P64, M75, L62, G51, T50, P46

45 127, W35, S33, V34, G32, T50, P46

V76, P64, T65, L62, G51, T50, P46

Epi#35

A24, L22, A23, S39, M12, 1107 50 A23, LH, A36, S39, M12 , 110 Epi#37

Y127, R123, L124, K97, N118 Y108, A23, LH, R123, Y127 5 A23, A24, LH, R123, Y127

Epi#39

A81, E57, H61, T65, P64, G60, L62

A81, E58, H61, T65, P64, G60, L62

10

Epi#40

R123, LH, A23, Y108, PHI, S91

R123, LH, A24, Y108, PHI, T113

15 Epi#41

PHI, Y108, L22, V112, S91 P114, Y108, L22, V112, S91

Epi#43 20 127, W35, A36, LH, Q37, S39, M12, 1107, T95

Epi#44

177, R86, P114, Y108, S91, V112 , PHI V120, Q116, P114, Y108, S91, V112 , PHI 25 L22, Q116, P114, Y108, S91, V112 , T113 L22, Q116, P114, Y108, A23, V112 , PHI

Epi#47

1129, Y127, E122, M119, R123, L124, N118, R86, P114 30 L133, Y127, E122, M119, R123, L124, N118, R86, P114

Epi#48

E122, Q116, P114, PHI, V112

S91, K88, P114, PHI, V112

35

Epi#50

HIO, Y6, W3, S2, T5

HIO, Y6, W3, T5, S39

40 Epi#51

K73, H68, K45, Q47, P46, S33, W35 QlOl, H30, D31, T50, K45, Q47, H68

45 Rag weed pollenδ:

Epi#03

L4, K37, A33, 134, Y17

L4, K37, A33, 134, Y29

50 Epi#05

A33, N36, T40, G3 , S20, L4

A33, N38, T40, G3 , S20, Y25

A33, N36, G3, T40, S20, 122

Epi#06

A33, N36, D2, C19, K24, P21

A33, N38, D2, S20, K24, P21 Epi#09

122, L4, D2, N38, DI, 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 , DI V27, K24, P21, L4, N36, D2 , G3 , DI

Epi#22

DI, D2, L4, K37 DI, D2, P21, K24

D2, L4, T40, DI

Epi#23

N10, E7, Y6, L4, P21

Epi#25

K32, 134, D30, V14

K37, 134, D30, V14

K16, 134, D30, V14

Epi#33

K32, Y17, V27, S20, K24

K16, Y6, P21, S20, K24 Epi#34

122, P21, S20, V27, G12 , Y17, T9

122, 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

Vesvδ :

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, 16, Y3

Epi#04

K106, Y107, S192, Q108, K7 , A105, 16

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#ll K179, 1176, Q177, V30, E178 K29, 1176, 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, 16, K5 , A105 K106, P191, D103, 16, 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, Qlll, 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, KIO

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

1176, 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, Qlll, 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

FO, A16, R102, W90, N25, Q95 FO, A16, R102, W90, N25, Q93

Betvl:

Epi#03

SAS: 270, Size 11.07: L24, K20, H76, 123, Y81

SAS: 204, Size 11.96: L24, K20, A16, 123, Y81 Epi#05

SAS: 298, Size 14.01: GllO, A106, A16, P14, Gill, TIO SAS: 242, Size 14.01: GllO, A106, A16, P14, Gill, T107

Epi#08 SAS: 464, Size 11.12: K123, E127, Gl , H121, F3

SAS: 455, Size 12.95: K129, E127, Gl , H121, F3

SAS: 438, Size 13.31: K123, D125, Gl , 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, GllO, V105, K80, A16, T77

SAS: 427, Size 20.55: D109, Gill, V105, K80, A16, T77 SAS: 398, Size 19.17: TIO, GllO, V105, K80, A16, T77 SAS: 381, Size 19.17: T10, Gill, 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, Gl, 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 1116, 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, 144, E42, E45 SAS 425, Size 12.68 R70, K54, 144, E42, E45 SAS 420, Size 14.01 R70, K55, 144, 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 8.70 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, GllO, TIO SAS 430 Size 19.17 T77, A16, A106, V12 , Gill, TIO SAS 392 Size 19.17 T77, A16, A106, V105, GllO, TIO SAS 391 Size 19.17 T77, A16, A106, V12, GllO, T107 SAS 375 Size 19.17 T77, A16, A106, V105, Gill, TIO

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 , TIO SAS 492 Size 19.04 123, R17, D156, Y150, S149, V12 , TIO 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, 144 Φ ϋ ns

C5 m

3 o o O rH

3 (ti a 4

H 0 U 4 α. <ϋ

A

4->

" -

CM

V r

r

90 C

L r

K77, D87, V76, H74, F75

Epi#09

L61, D64, 168, H74, F75, T70, N71 N114, N46, D113, K48, N71, T70, T49 G83, N46, D113, K48, N71, T70, T49

Epi#10

L40, 113, D42, N44, V81, K48, N46, N114, G115 L40, 113, D42, N44, V81, K82, N46, N114, G115

L37, D19, G20, V18, V3 , D4 , K6 , A120, T107, V105

Epi#ll

F75, K51, llll, Q45, V116, D113 F75, K51, llll, Q45, V81, D113

Epi#12 Y90, E38 Epi#13

H30, R31, P95, A98, P99, SlOl, G60, L61

Epi#15

K96, P99, D129, 128, R128, A98 K96, P99, D129, 1127, R128, A98

K96, P99, D129, 129, R128, A98

K55, P66, D64, 168, T70, G67

Epi#18 R31, R128, 128, G125, T123, H124, V105 R31, R128, 1127, G125, T123, H124, V105

Epi#22

DI, M17, D4, V3, K6 DI, M17, D19, P34, K96

DI, M17, D4, V5, K6

Epi#23

K14, Nil, E12, N44, Q85, P79 K14, Nil, E12, NIO, Q45, P79

K14, Nil, E12, N44, Q84, P79

K14, Nil, 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 o o

3 a

H U α.

co

CM

90

Epi#41

P34, L37, V106, S57

Epi#42 P26, S24, G125, R128, R31 P99, SlOl, 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, 113, Y86 L17, K89, A72, 188, Y90

L17, K89, A72, 152, Y90

Epi#04

K15, SI, Q2, K14, V16, L17 K15, SI, Q2, K14, A39, L17 K15, SI, Q2, K14, V40, 113

Epi#05

G60, A56, L61, P99, G32, R31, H30, 197 G60, A56, L61, P99, G32, R31, H30, 128

Epi#06

Epi#08

K55, D64, S57, V106, F35

K55, E62, S57, V106, F35

Epi#09

L61, G60, E102, R128, 128, K126, N103, T123, V105

L61, G60, E102, R128, 1127, K100, N103, T123, V105

L61, G60, E102, R128, 1127, 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#ll

K14, 113, Q85, V81, E42

K15, 113, Q85, V81, E42

K14, 113, 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, 168, K89, A72

K55, P66, D69, 168, K89, A39

K55, P66, D64, 154, K109, G115

K55, P66, D64, 154, K109, A9

Epi#18

R31, 129, A125, SlOl, E102, N103

R31, 129, A125, SlOl, E102, V104

R31, 129, A125, T123, A122, V105 C5 o o

3 a

H U α.

IΛ

90

^H o o

P19, V3, L17, K89, D87 T123, V104, L61, K100, N103

Epi#38

5 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

10

Epi#40

G60, L61, A56, T107, K6 , T123

A39, L17, G20, T123, P26, S24

G60, L61, A56, T107, K55, S57

15 G60, L61, A56, T123, K126, SlOl

Epi#41

P19, L17, V3, SI

P19, L17, V5, S24

20

Epi#44

V65, D64, P66, W92, Y90, A39, V18, P19

L61, D64, P66, W92 , Y90, A39, V18, T91

25 Epi#45

R31, P34, F35, N93 , V94

K96, P34, F35, N93, G32

Epi#47 30 1127, SlOl, R31, 197, A98, L61, N103, P99, P95 128, SlOl, R31, 197, A98, L61, N103, P99, S57

Epi#48

H30, K96, P95, P99, G60

35 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

40 H30, K96, P95, P34, G32

Q36, K89, P70, P66, V65

Epi#50

D69, Y90, W92, P66, P70

45 D69, Y90, W92, P34, P95

D69, Y90, W92, T91, P34

D69, Y90, W92, V94 , P95

D69, Y90, W92, L37, P19 so Epi#51 K126, H124, E102, R128, 128, R31, H30 T123, H124, K126, R128, 128, R31, H30 D4, H124, K126, R128, 128, R31, H30 Phlp2:

Epi#02

T87, K85, Q61, S38, R34, R67 T87, K85, Q61, P63, R34, V42

Epi#03

KIO, A90, 188, Y86

KIO, A18, 188, Y86 Epi#04

R34, S38, Q61, K85, T87, 188 R34, S38, Q61, K85, T87, A90

Epi#05 G47, A18, S12, T87, G89, T91, T5 , VI

G73, A29, L69, T27, G50, T53, T45, V42

GH, A18, L20, T91, G89, A90, T87, 188

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

KIO, E9, GH, A18, H16, F54 K46, E48, G47, A18, H16, F54 KIO, 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, GH 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 , VI 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

KIO, N13, E14, L60, Q61, P59 KIO, N13, E14, L60, Q61, P83

KIO, 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

188, 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

188, N13, S12, H16, K15, P59, L60

188, N13, S56, H16, K15, L60, P59 188, N13, A18, H16, K15, P59, L60

Epi#33

K46, F54, V42, S56, K15 H16, F54, V42, S56, K15

Epi#34

VI, P2, T5, V4, P94, Y92, T87 VI, P2, T5, L20, G89, T91, T87 V81, P94, T5, VI, 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, KIO, S12

G49, L20, G89, T87, KIO, T7

Ξpi#44

V77, R67, D79, P94, Y92, A93, VI, P2 L69, R67, D79, P94, Y92 , A93, VI, T5

Epi#45

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, Ξ40, A43, R34, L60, N13, P59, S56 S38, G37, E40, A43 , R34, L60, N13, P59, S56

Epi#48

E24, K3, P94, P2 , VI

E84, D80, P94, P2 , VI Epi#50

D39, W41, A43, T45

D39, W41, V42, T45 Epi#51

D79, H36, E84, T87, KIO, GH , H16

D39, H36, Q61, K85, P63, R34, W41

D79, H36, E40, D39, G37, R34, W41

Q61, H36, E84, T87, KIO, GH , 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' (lsup.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 para- graph "Methods" above and the program can be found in Appendixes .

The critical parameters used in this screening included: i) a maximal distance between the alfa-carbon atoms of subsequent amino acids, ii) a minimal accessability of the amino acid of

20A2, iii) the largest maximal distance between the most distinct amino acids should be less than 25A iv) the best epitope were taken, v) the homology with the epitope pattern of interest was 100%

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 IB) .

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, 1288, Y289 L66, K61, H254, 1253, Y329

Epi#04

R122, Y175, S184, Q172, Y169, A454, 1455

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, Vlll, F49 A24, D25, S30, Vlll, 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#ll F49, F109, 191, 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, 1288, T290, G294

R160, P157, D153, 1154, T462, G90

R286, P307, D283, 1288, 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

5 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

10 L295, G294, L291, R286, E299

1288, K404, L291, R286, E299

L348, K352, L354, F380, E299

Epi#33

15 K352, Y355, V374, S371, S365, K337

K352, Y355, V374, S365, S340, K337

Epi#34

V463, W466, S468, V470, P467, T464, T462

20 1469, W466, S468, V470, P467, T464, T462

1154, W466, S468, V470, P467, T464, T462

V463, W466, S468, V470, P467, S465, T464

Epi#37

25 T362, A359, L348, K352, D357

T360, V346, L348, K352, D357

T362, A359, L348, K352, D349

Epi#38

30 G438, E259, A435, R68, L66, N69, P434, S431

Epi#39

A353, E299, R286, P307, G243, L234

A300, E299, R286, P307, G243, L234 35

Epi#40

A205, L143, G146, Y147, P467, T464

G146, L143, A205, T204, A201, S209

A451, A450, T448, P446, S444

40

Epi#41

P467, Y147, L143, V206, S149

Epi#42 45 L66, P434, S431, N430, R429, R428

L104, P123, S95, G101, P94, R122, R125

L104, P107, S95, G96, P123, R125, Q172

Epi#44

50 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, NHO, D112, G31 R413, P41, F49, Y50, NHO, D33, G31 D44, P46, F49, Y50, NHO, D112, G31

Epi#4 6

Y175, R125 P123, G174 Q172

Y169, R125 P123, G174 Q172

V432, R429 P434, N69, G70

Y175, R125 P94, N93, G90

Y175, R122 P123, N182 G121

Y175, R125 P94, G101, A102

Y175, R125 P94, G118, A115

Y175, R125 P94, G101, G96

Y175, R122 P123, N182 G183

Epi#48

S211, D214 P446, G447

E259, K337 P434, V432

S215, D214 P446, G447

S209, D214 P446, V445

E259, K337 P434, V433

Epi#50

R122, Y175 T117, S119

R125, Y175 S119, T117

Epi#51

T390, H391 Q409, R413, S411, W317

T390, H391 S405, 1288, K404, W317

D406, H391 Q409, R413, S411, W317

T390, H391 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, 1108, Y104 K136, A137, 1108, Y104 K136, A134, 1108, 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, Al, N77, P40, G211, S38, S37, V44

G80, Al, 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#ll

K136, 1108, Q103, V51, D98

Epi#12 Y171, E195

Epi#13 SlOl, W106, P52, T55, A48, P56, S49, G47, F58 S105, W106, P52, T55, A48, P56, S49, G47, W113

Epi#15

N25, P239, D120, 1115, K141, A144 N240, P239, D120, 1115, 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

Epil9

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, 1115, D120, N25 K141, 1115, D120, N118

K141, 1115, 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, GlOO, D61

A99, D61, Q59, F58, E54, L96, Q103, SlOl, D98

Epi#29

G102, Q103, L96, E54 GlOO, Q103, L96, E54

Epi#30

179, N76, S87, H17, S18, P14, V4

179, N76, S87, H17, Q19, P14 , V4 Epi#31

L257, Q185, N184, R186, F189, V203, 1205, D181

L267, Q10, N184, R186, F189, V203, 1205, 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 1115, W113, M50, V51, P52, T55, S53 1108, W106, S105, V51, P52, T55, S53 Epi#35

A99, L96, S49, M50, 1108 A99, L96, S49, M50, 1107

Epi#36 A137, A134, A133, G131, Y104, S105, Q103, V51, A48, W113 A134, A137, A133, G131, Y104, SlOl, 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

5182, Q185, D181, Y6 , S9, V4 , P14

5183, 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, 138, Y54 K13, A43, 138, Y54

Epi#04 R153, S151, Q145, Y147, R146, 1131

R153, S151, Q145, Y147, R146, G144

R153, S151, Q145, Y147, R146, L142

Epi#05 G3, Al, S183, T95, G101, A100, S96, G97 C5 o o

3 a

H U α. r- r- VO

CD CM

90

Al, S183, R4, S5

Epi#18

N118, R4, S181, , G3 , , S117, L115, , A78, S80

5 N34, N32, R37, F35, , A33, Y54 , S45, , , A43, V52

Epi#19

D157, N154, S151, Q145, R146, L142

D178, N176, S151, Q145, R146, G144

10

Epi#22

D40, A43, D42, W18, K20

D40, A43, D42, A19, K20

15 Epi#23

R158, N154, E155, L142, Q145, P143 R153, N154, E155, S151, Q145, P143

Epi#24 20 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

25 Epi#25

R158, K164, W169, D172, N176 R4, H119, 177, E82, N81

Epi#26 30 W18, S15, E82, W85, P23, A19, D42 W18, S15, E82, W85, P23, G84, D203

Epi#28

1131, D133, Q138, L142, E155, K164, F159, P165, D161

35 1131, D133, Q138, L142, E155, K164, F159, P143, R158

1131, D133, Q138, L142, E155, K164, F159, P160, R158

Epi#29

1131, R146, L142, R158, E155 40 G144, Q145, L142, R158, E155

Epi#30

G79, N81, A78, H119, S117, 177, L115

G79, N81, A78, H119, S76, 177, L115

45

Epi#31

L142, R158, N154, R153, W169, F171, D172

Epi#33 50 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, Till, A74, T6

G79, L115, G113, Till, A74 , S15

G79, L115, G113, Till, A74, SHO

G116, L115, G113, Till, A74, T6 G79, L115, G113, Till, 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

1131, 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, 179

Epi#06

G211, N61, D97, R98, S53 , P55 G102, N99, D97, R98, S53, P55 GlOO, 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, 144, R43, A45

N24, P86, D89, 144, R43, G46 N76, P86, D89, 144, R43, A45

N24, P86, D89, 144, 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

121, N18, Q15, Q275, R19, G20 121, N18, Q15, Q275, R237, G20 E197, N265, S261, Q161, R160, G162 E197, N265, S261, Q161, R160, G157 121, 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, 144, D89, N24 R27, H120, 1115, E112, N116 Epi#28

L104, Q109, 1115, E112, W113, F50, S53, R98

A105, Q109, 1115, E112, W113 , F50, G102, R98

A108, Q109, 1115, E112, W113 , F50, S53, R98

V107, Q109, 1115, E112, W113, F50, S53, R98

Epi#29

1147, N140, L137, R141, E136 G146, N140, L137, R141, E112 1115, N143, L137, R141, E136 G102, N99, L96, R98, E54

Epi#30

G211, N212, S38, H59, S57, 151, P55 G211, N61, S57, H59, S38, P40, L75 G211, N212, S38, H59, S49, 151, P55 G211, N212, S38, H59, P55, 151, 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

179, P40, S38, G211, R213, Y209, S216

179, P40, S38, G211, R213, Y214, T210 151, 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, 1186, 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 C5 o o

3 a

H U ID cn cn α. O CO CO

CM CM CM

P £ ro ∞ o vo co cn rH CM CM

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CM

90

CO co co co co ro ro ro ro ro

H H rH H H

P P P P P

o

I-- CM

LD ro

^■Φ pq O ro ro rf ro r ro i- CM ro ro 0 σ

^■φ ^•φ ro CD

G412, N304, A306, H309, 1312, P314, V419 1312, 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, 1454, 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, 1358

G372, L369, A362, S360, M359, 1358 A365, L369, A362, S360, M359, 1358

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

Al, 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

5 Epi#42

L392, P395, S428, G430, P388, R330, Q332

Epi#44

S360, Q363, D367, P370, Y347, A371, G372, T345 io 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

15 R409, P322, F418, Y416, N420, D313, V419

K423, P314, F418, Y416, N420, D313, V419

R175, P180, F21, Y176, R266, D166, G268

Epi#46 20 A296, R259, R234, P300, N299, A301 Y256, R259, R234, P300, N299, Q302

Epi#47

1212, S211, R234, L303, A301, N299, P300, P298 25 1212, S211, R234, V232, A301, N299, P300, P298

Epi#48

S158, Q160, P157, P155, V504

S499, Q501, P55, P155, V504

30 E488, Q485, P480, P479, V481

Epi#49

D367, L369, V352, P350, Q357, Q363, M359, N478 D367, L369, P370, P350, Q357, Q363, M359, N478 35

Epi#50

D291, Y256, W257, S295, P298

D254, Y256, W257, T293, S295

40 Epi#51

D307, H309, E228, T218, P229, T231, H230

R234, H215, E216, T231, P229, H230, H309

D248, H215, E216, T231, P229, H230, H309

45 Epi#52

F69, A100, T98, R71, W75, T73, Q70 F97, A100, T98, R71, W75, T73 , Q70

50 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, 1404, Y398

L307, K305, H401, 1404, Y398

L307, K305, A304, 1404, Y398 Epi#04

R167, S166, Q168, R172 , N171, 1173

R177, Y131, S128, Q125, R123, N124, 1127

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#ll

F169, 1173, Q170, D162 L195, 1173, Q170, D162 Epi#12

Y192, E188

Y357, E354

Epi#13

H12, L13, P369, A375, P374, S372, P330, Wll

H12, L13, P369, A375, P374, S372, P330, L334

H12, L13, P369, A375, P374, S372, P330, G331

Epi#15

N451, P453, D447, 1448, T449, A378

N451, P453, D447, 1448, 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 1173, 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 1448, P453 G454, N451 H446 K478 1448, P453

Epi#31 Q168, N171 R172 W163 M196, 1173, D162 Q170, N171 R172 W163 V161, 1173, 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 LI14 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, TllO

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, Wll, Y362, A375, V373, T370 S333, D327, P330, Wll, 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, 185, R24, A87, N90, R31, S28

A250, G252, E254, N249, R248, F256, N279, R241, S238

Epi#48

S372, H371, P374, P369, V373

Epi#49 D51, Wll, L13, V52, P14, Q18, Q80, T77, N21 D51, Wll, 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, 153, Y56 L99, K51, A49, 153, Y43

Epi#04

R28, S331, Q333, K97, R50, 153 R28, S331, Q333, K97, R50, A49

Epi#05

G108, A106, N107, GllO, S109, SHI, 159

GllO, A106, N107, G108, S109, SHI, L112 G108, A106, N107, GllO, SHI, S117, Y121

G108, A106, N107, GllO, SHI, S109, G135

GllO, A106, L68, P214, G217, S219, Y220

G108, A106, N107, GllO, SHI, 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, 153, K54, N64 , N63, R61

N44, A49, R50, 153, 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, 187, T83, G86

N215, P46, D82, 187, T83, G86 Epi#18

N216, N44, R50, 153, A49, P46, N215

N215, N44, R50, 153, 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, Sill, 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

1259, D257, Q254, E253, K267, F266, S260, R189 1259, D257, Q254, E253, K267, F266, S260, K258

Epi#29 L68, G108, L134, F170, E137 G135, N163, L134, F170, E137

Epi#30

GllO, N107, A106, H71, L68, L104, L112 G108, N107, A106, H71, L68, P214, V213

GllO, N107, A106, H71, P214, L68, L104

GllO, N107, A106, H71, L68, L104, L134

Epi#33 Q12, Y220, V207, S222, S192, R189 K190, F193, V207, S222, S192, R189 Q16, Y13, V207, S222, S192, R189

Epi#34 V26, WI, T27, G29, R28, S331, T332 WI, P21, T27, V26, R278, Y279, T255

Epi#35

G135, L134, S225, M221, 1209 GllO, L134, S225, M221, 1209 G108, L134, S225, M221, 1209 G162, L134, S225, M221, 1209

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, LI12 , 1115, 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, RIO, P14, R19, R269

T249, N263, S9, RIO, P14, R19, R269

Epi#03 K27, A86, 143, 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#ll

F49, L94, 1105, Q107, V102, E134

F49, K92, 1105, 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

1240, W235, S234, Q239, R241, K245 D175, N179, S182, Q185, R180, G252

Epi#23

R143, N114, EHO, S139, Q135, A131 R143, N115, EHO, 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

177, N74, L41, R44, E87 V4, N74, L41, R4 , 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, RIO, W6, V197, D175 L251, R180, N178, RIO, 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 5 A131, E134, L133, T132, P129, G125, L124 A166, E134, L133, T132, P129, G125, L124

Epi#40

R44, L41, G78, T207, P39, T37 io R19, L21, G20, T22, K231, S234

R180, L256, G252, T254, A188, S158

Epi#41

P127, Y161, L133, V102, S99

15 P127, Y161, L133, V102, S103

P127, Y161, L133, V102, SlOl

P127, Y161, L133, V102, S126

Epi#42 20 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 25 105, Will, A47, G46, Q57, S36, L41, 143, T37

Epi#44

S126, R164, P127, Y161, S158, A188, T254

S128, R164, P129, Y161, S158, A188, T254

30

Epi#46

A15, R269, R19, P14, N18, G20

A266, R269, R19, P14, N18, A15

35 Epi#48

S55, Q57, P54, P51, G52 E53, Q57, P54, P51, G52

Epi#50 40 RIO, W6, S3, S76

R241, W235, S234, P233 RIO, W6, V4, S9

Epi#51 45 Q239, H243, T247, R269, R19, K231, W235 R19, H17, E265, R269, K231, S234, W235

Epi#52

A15, S9, RIO, W6, N198, Q176 A15 , S9 , RI O , 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 1410 Y404 L313, K311 H407 1410 Y404

Epi#04 R171, S170 Q172 R176 N175, 1177 R181, Y135 S132 Q129 R127, N128, 1131

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#ll F173, 1177 Q174 D166 L201, 1177 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, 1454, K458, G456

K458, P459, D453, 1454, T455, A384

N457, P459, D453, 1454, 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, 189, 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, 1454, E391, N36 R485, K484, 1454, E391, N395

Epi#26 W167, S17 W140, V117, G182, D183 W167, S17 W140, V165, Ξ121, D134 W167, S17 W140, V165, E121, E130

Epi#27 E212, E21 K156 E216, E21 K242

Epi#28 L201, D16 W167, E138, W140, Q169, S170, R171 L201, D16 W140, E138, W167, F173, S170, R171 L201, D16 W167, E138, W140, Q169, S170, R171

Epi#29 V214, N21 R219, E222 G96, H90, 32, E86 V214, R21 R218, E212

Epi#30 G456, N45 K484, 1454, P459 G362, M32 H324, K320, P322, V318 G362, M323 H321, K320, P322, V318 G460, N45 K484, 1454, P459

Epi#31 L217, R21 R218, F245, V214, D248 L217, R21 R218, F245, M208, D209

Epi#33 K383, Y372 S378, A381, K385 K383, Y372 S378, A381, K38

Epi#34 W439, W46 V450, R485, T461, P459 W439, W46 V462, R485, T461, P459 Epi#37

T251, R21 R219, N215 P211, V214 R219, N215 A256, R21 R219, N215 Epi#38

G435, E437 P434, S478 G436, E43 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

5 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 io L173, K172, S170, T136, Y135, R118, R181

L173, R171, S170, T136, Y135, R118, R181

L390, K395, Q394, R393, T5, Y398, R400, R415

Epi#03 15 K438, H407, 1410, Y404

Epi#04

K172, S170, Q169, R171, N174, L173 R171, S170, Q169, K172, N175, 1177 20

Epi#05

G456, A459, R458, T461, G460, T452, T463, V450

G456, A459, R458, T452, G460, T461, T463, G448

25 Epi#06

A51, N54, D20, R76, Q71, P146 G73, A51, D55, S52, K72, P146

Epi#07 30 G456, T455, S384, D387, R393, P388, D453

Epi#08

K259, S255, V222, H252, F245

K259, G258, A256, H252, F245

35

Epi#09

N128, V131, R176, D166, K172, N175, N174, R171

Epi#10 40 467, N445, R444, F441, R415, R400 D467, A466, R444, F441, R415, R400

Epi#ll

F69, K72, 175, Q53 , V56, D55

45

Epi#12 Y16, E337 Y363, E360 Y198, E194

50 Epi#15

K385, P388, D453, 1454, R458, A459 K385, P388, D387, 1454, ^T452, A459 K385, P388, D387, 1454, R458, G456 5

Epi#17

A87, S29, R28, S32

A91, S29, R28, S32

N445, R444, A466, T463, T461, N471, N437 N445, R444, A466, T463, T461, T452, V450

Epi#19 15 166, W167, S170, Q169, R171, K172 E138, W167, S170, Q169, R171, K172 E134, T136, S170, Q169, R171, K172

Epi#22 20 D209, P211, D207, Y160, D183

Epi#23

R400, N418, E416, N445, Q449, A466

R82, N83, E68, N70, F69, P146

25

Epi#24

E134, G133, E130, P124, R176, L173, K172

E134, K179, E130, P124, R176, L173, K172

30 Epi#25

R444, K446, W469, D467, N445

R171, K172, W167, D166, N175

R171, K172, W167, D166, N174

35 Epi#26

W167, S170, E138, W140, V165, E121, E130

W167, S170, E138, W140, V165, E121, E134

W167, S170, E138, W140, V165, E121, D166

40 Epi#27

E130, E121, D166, K172 D36, E391, D387, K385 E134, E121, D166, K172

45 Epi#28

L201, D166, Q169, W140, E138, K172, S170, R171 L173, D166, Q169, K172, E138, W167, S170, R171

Epi#29 50 V131, R176, L173, R171, E138 1177, N175, L173, R171, E138 1177, N174, L173, R171, E138

Epi#30 139, 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, 1177, D166

L173, R171, N174, R176, W167, V165, 1177, 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#47 175, 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, RIO, P14, R19, R275 T255, N269, S9, RIO, P14, R19, R275

Epi#03 K27, A88, 144, 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 GlOO, 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

1246, 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

5 Epi#29

V244, Q245, L148, R145, E112 V244, N173, L148, R145, E112

Epi#30 10 G61, N62, A98, H64, L96, P52, P55 G20, N18, A15, H17, S87, L75, P40 179, N76, S87, H17, Q12 , P14 , V4 GlOO, N62, A98, H64, L96, P52, P55

15 Epi#31

L262, R186, N184, RIO, W6 , V203, D181 L257, R186, N184, RIO, W6 , V203, D181

Epi#33 20 Q109, F50, P52, S49, S56, K94 Q109, F50, P55, S56, A48, K94

Epi#34

W241, P239, S242, G146, R145, S141, S144

25 1165, P194, T260, G258, R186, S188, S156

V104, P129, S130, G127, G102, SlOl, S99

V244, W241, S242, G146, R145, S141, S144

1165, P194, S170, G127, P129, S130, S103

30 Epi#37

P14, A15, L21, R19, N18 T143, R145, L148, R247, N252 T143, V244, L148, R145, N116

35 Epi#38

L96, G97, E54, A98, N62, P210, S212 L96, G97, E54, A98, N62 , P210, S37

Epi#39 40 A15, E271, H17, R19, P14, G20, L21 A254, E271, H17, R19, P14, G20, L21 A272, E271, H17, R19, P14, G20, L21

Epi#40 45 R186, L257, G258, T260, P194, S162 R186, L262, G161, Y192, P194, T260

Epi#41

P194, Y192, L262, S259 50 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, 144, 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 RIO, W6, S3, S78

RIO, W6, V4, S9

RIO, 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, RIO, W6, N204, Q206 A15, S9, RIO, 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, 1165, Y167 L196, K170, A194, 1165, 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, SlOl, S103, Y104

Epi#06

A24, N25, D120, 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, GlOO, 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, D181, 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 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 SAS 309, Size 13 46 P127, Yiei, 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, GlOO, S99, L124

Epi#19 SAS: 295, Size 15.06: D175, W6, S9, Q12, RIO

SAS: 278, Size 21.23: EHO, T141, S236, Q239, R241

Epi#23

SAS 486, Size 19.88 R143, N114, EHO, 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, Will, EHO, Q135, S139, R143

SAS: 492, Size 24.70: V102, Q107, F49, E53, Q57, G46, R44

SAS: 480, Size 22.76: V50, Q107, Will, EHO, Q135, S139, R143

SAS: 452, Size 19.08: V50, Q107, F49, E53, Q57, G46, R44 SAS: 441, Size 24.70: V102, Q107, EHO, Will, 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 14, 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, RIO, W6, V197, D211

SAS 352, Size 22.95 L251, R180, N178, RIO, W6 , V197, D211 SAS 350, Size 21.62 L256, R180, N178, RIO, W6, V197, D175 SAS: 339, Size 17.75: L251, R180, N178, RIO, 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

Epi#19

SAS: 293, Size 15 04 D175, W6, S9, Q12, RIO

SAS: 291, Size 17 13 D191, N242, S236, Q239, R241

SAS: 273, Size 21 24 EHO, T141, S236, Q239, R241

Epi#23 SAS: 463, Size 19.84: R143, N114, EHO, 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, EHO, 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, RIO, W6 , V197, D175

SAS: 342, Size 17 75 L251, R180, N178, RIO, 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 3 38822,, SSiizzee 2233. 3333: 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, N13I 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, RIO

SAS: 276, Size 21.28: EHO, T141, S236, Q239, R241 Epi#23 SAS 497, Size 19 86: R143, N114, EHO, 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, EHO, S139, Q135, A131

Epi#28

SAS 554, Size 22.17 AlOl, Q107, 1102, E134, Q135, S139, R143

SAS 532, Size 19.36 1102, Q107, Will, EHO, Q135, S139, R143 SAS 527, Size 22.79 V50, Q107, 1102, E134, Q135, S139, R143

SAS 509, Size 24.76 1102, Q107, F49, E53, Q57, G46, R44

SAS: 508, Size 22.17: AlOl, Q107, Will, EHO, Q135, S139, R143

Epi#31 SAS: 355, Size 21.56: L256, R180, N178, RIO, W6, V197, D175

SAS: 352, Size 17.71: L251, R180, N178, RIO, 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_τ £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 Ξpi#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, EHO, 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, EHO, S139, Q135, A131 SAS 446 Size 19.99 R143, N138, E134, S130, Q135, A131

Epi#28 SAS: 505 Size 19.43 1102, Q107, Will, EHO, Q135, S139, R143 SAS 500 Size 22.22 AlOl, Q107, Will, EHO, Q135, S139, R143 SAS 499 Size 24.79 1102, Q107, F49, E53, Q57, G46, R44 SAS 494 Size 24.56 AlOl, Q107, F49, E53, Q57, G46, R44 SAS 441 Size 24.79 1102, Q107, EHO, Will, F49, G46, R44 Epi#29 SAS: 216 Size 9.94: 143, 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: 177, 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, RIO, W6, V197, D175 SAS: 370 Size 17.72: L251, R180, N178, RIO, 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*f37

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*_r39

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

Epϋ_r40

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, 143, 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*_r12

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, Ξ134, 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: EHO, T141, S236, Q239, R241 Epi#23

SAS 491, Size 19.86 R143, N114, EHO, S139, Q135, A131

SAS 482, Size 15.76 R164, N167, E134, S139, Q135, A131

SAS 465, Size 22.69 R143, N115, EHO, S139, Q135, A131

SAS 462, Size 18.17 R143, N138, E134, S139, Q135, A131 SAS 439, Size 18.17 R143, N138, EHO, S139, Q135, A131 Epi#28

SAS 445, Size 22.79: V50, Q107, Will, EHO, Q135, S139, R143

SAS 426, Size 19.06: V50, Q107, F49, E53, Q57, G46, R44 SAS 370, Size 19.06: V50, Q107, EHO, Will, F49, G46, R44

Epi#31

SAS: 347, Size 21.62: L256, R180, N178, RIO, W6 , V197, D175

SAS: 339, Size 17.74: L251, R180, N178, RIO, 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 4 46611,, S Siizzee 1177. 2255: G158, A189, R165, P128, G126, S129

SAS 4 43399,, S Siizzee 1177. 2222: G158, A189, R165, P128, G126, S127

SAS 4 43366,, S Siizzee 1177. 2255: G158, A189, S159, P128, G126, S129

SAS 4 42200,, S Siizzee 1177. .2255: 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}_r12

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: Epi* H8

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: Elll, 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, Elll, 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 , Si ze 13 . 93 : R45 , K27 , D119 , E88

Epi#2 8 SAS 502, Size 19 99 V103, Q108, W112, Elll, 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, Elll, Q136, S140, R144 SAS 439, Size 19 16 V51, Q108, F50, E54, Q58, G47, R45

Epi#3

SAS: 354, Size 21.73: L257, R181, N179, RIO, W6 , V198, D176

SAS: 348, Size 17.85: L252, R181, N179, RIO, 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 21.81 144, R45, Y90, A48, V51, P52

SAS: 386, Size 19.16 144, 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, 178, S3, W6

SAS: 485, Size 21.66: L21, R19, H17, D75, Q2 , 178, 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 suf ce area of the epitope in A2.

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' (Figure 1) .

The data in Figure 1 show a significant overlap between the ar- eas 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.

Table 10: Experimental conditions for evaluation of Subtilisin variants Q12D.

Table 11: Experimental conditions for evaluation of Subtilisin variants Q12D.

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

Water hardness was adjusted by adding CaCl₂ and gCl₂ (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 were evaluated by calculating a performance factor:

Variant Blank

Savinase Blank

P : Performance factor

^Variant ^: Reflectance of test material washed with variant

® R-_Savinase ^: Reflectance of test material washed with Savinase

^RBlank^: 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 : Subtil isin variants and improvement classes .

Improvement Variants class

C I44V, Q12D

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

Appendix A Source code for the core C program (epitope. c

/* This is epitope. c */ /* EPF 25-10-2000 */

DEFINES

#define MAXRESIDUES 1000 #define MAXCONSENSUS 15 #define MAXEPITOPERES 30000 #define MAXEPITOPES 10000 ttdefine AMINOACIDS "ACDEFGHIKLMNPQRSTVWY" #define AMINOACIDS3 "ALA CYS ASP GLU PHE GLY HIS ILE LYS LEU MET ASN PRO GLN ARG SER THR VAL TRP TYR " #define REVISIONDATE "12-02-2001" #define ma (A, B) ((A) > (B) ? (A) (B)) #define min (A, B) ((A) < (B) ? (A) (B))

/" INCLUDES

#include <stdio.h> #include <stdlib.h> #include <string.h> #include <math.h> #include <limits.h>

/* STRUCTS struct residue { char ltr3 [3] ; char ltr; float x, y, z; int sasa, number,- int member_of_epitopes; /* how many epitopes is this residue part of ? */

}^; struct epitoperesidue

{ int parent; /* -1 if top level */ int residue; /* -1 if gap */ char level ;

struct epitope

{ int sasa, gaps, residues, res [MAXCONSENSUS] ; char epi [255] ; char subset; /* is this epitope a subset of another */ float size;

};

/* GLOBALS

struct residue res [MAXRESIDUES] ; struct epitoperesidue epires [MAXEPITOPERES] ; char consensus [MAXCONSENSUS] [22] ; struct epitope epi [MAXEPITOPES] ; int numofres = 0, numofepires = 0, consensuslength = 0; int minsasa = 0, numofepitopes = 0, numofsubsets = 0; float mindist = 7, sqmindist, maxsize, sqmaxsize, minlength = 0; int maxepi = 0, minlength_residues, longestepitope;

/* _FI_LE FUNCTIONS */

int readconsensus (char *filename) {

/* return length of consensus sequence */ int i = 0 ; FILE *infile; char buffer [255], end = 0 ;

if (infile = fopen (filename, "r"))

{ /* This code adds linefeeds to the consensus file. This is because there must be a newline after the last line. Because of permission problems, this has been moved to the wrapping cgi-script instead fclose (infile) ; infile = fopen(filename, "a"); fprintf (infile, "\n\n") ,- fclose (infile) ; infile = fopen (filename, "r"); */ while (! feof (infile) && lend)

{ fgets (buffer, 255, infile) ; if (strlen (buffer) > 22)

{ printf ("Too many residue types in consensus residue %d\n",i+l); printf ("using all 20 types instead. \n") ; strcpy (consensus [i] , AMINOACIDS) ; } else if (strchr (buffer, '*') ) /* wildcard '*' means any residue, but no gap

*/ strcpy (consensus [i] , AMINOACIDS); else if (strchr (buffer, '?') ) /* wildcard '*' means any residue or gap */ { strcpy (consensus [i] , AMINOACIDS); strcat (consensus [i] , "-");

} else if ( ! strpbrk (buffer, "ACDEFGHIKLMNPQRSTV Y*?") ) /* empty line, end the loop */

{ end = 1; i--;

} else strncpy (consensus [i] , buffer, strlen (buffer) -1) ; i++; } } fclose (infile) ; consensuslength = i; return i ;

int readpdbCA(char *filename) /* return number of residues */ int i = 0 ,- char *j ,-

FILE *infile; char buffer [255] ; char aminoacids [20] = AMINOACIDS; char aminoacids3 [80] = AMINOACIDS3 ; if (infile = fopen (filename, "r"))

{ while ( Ifeof (infile) )

{ fgets (buffer, 255, infile); if (Istrncmp (buffer, "ATOM", 4) && ! strncmp (buffer+13 , "CA" , 2) ) /* get only the CA atoms */

{ strncpy (res [i] . Itr3 ,buffer+17, 3) ; if (j = strstr (aminoacids3 , res [i] .ltr3) ) res [i] . Itr = aminoacids [ (j -aminoacids3) /4] ; else

{ printf ( "Unknown residue type: %s\n" , res [i] . Itr3) ,- res[i] .Itr = 'X' ;

} res [i] . x = atof (buf fer+30 ) res [i] . y = atof (buf fer+38 ) res [i] . z = atof (buf fer+46 ) res [i] . member_of_epi topes = 0 ; res [i] . number = atoi (buf fer+22 ) ; i++ ; } } } numo res = i ; return i ; }

int readdssp(char *filename)

{

/* return number of residues */ int i = 0; char *j ; FILE *infile; char buffer [255] ; strcpy (buffer," "); if (infile = fopen( filename, "r"))

{ while ( Ifeof (infile) && strncmp (buffer, " # RESIDUE AA" ,15)) /* find where data begins */ fgets (buffer, 255, infile); while ( Ifeof (infile) )

{ fgets (buffer, 255, infile); if (Ifeof (infile))

{ if ((buffer[13] == res [i]. Itr && atoi (buffer+5) == res [i] .number ) I I (strchr ("abcdefghij klmnopqrstuvwxyz ", buffer [13] ) && res [i]. Itr == 'C && atoi (buffer+5) == res [i] .number ) )

{ res [i] . sasa = atoi (buffer+35) ; i++;

} else printf ("Inconsistency between pdb and dssp file at residue %c%d\n" ,res [i] .Itr, res[i] .number) ; } } } if (i != numofres) printf ("Inconsistency between pdb and dssp file: wrong # of residues (%d) in pdb, (%d) in dssp\n", numofres, i) ; return i;

}

void writedatafile (char *filename) { int i;

FILE *outfile;

if (outfile = fopen(filename, "w")) { fprintf (outfile, "# seq pdb AA epitopes\n" } ; fprintf (outfile, "# seq \n"); for (i=0; i<numofres; i++) fprintf (outfile, "%4d %4d %c %4d\n",i+l , res [i] .number, res[i].ltr, res[i] .member_of_epitopes) ; fclose (outfile) ; } }

/* ANALYSIS FUNCTIONS */ int addchild(int parent, int residue, char level) { if (numofepires == MAXEPITOPERES)

{ printf ("Sorry, program constant MAXEPITOPERES exceeded, increase and recompile program\n") ; exit (0) ; } epires [numofepires] .parent = parent; /* should be -1 for the top level */ epires [numofepires] .residue = residue; /* should be -1 for a gap */ epires [numofepires] .level = level; numofepires++;

/* if (numofepires % 10 == 0) printf ("Added %d epires\n" ,numofepires) ; */ return numofepires; } float sqdist(int i, int j)

{

/* returns the square of the distance between the coordinates for residues i and j */ return (res [i] .x-res [j ] .x) * {res [i] .x-res [j ] .x) + (res [i] .y-res [j ] .y) * (res [i] .y- res [j ] .y) + (res [i] . z-res [j ] . z) * (res [i] . z-res [j ] . z) ; }

void f indepitopes (void) /* This is the core algorithm */

{ int i, j, k, nogapanchestor;

/* Find parents */ for(i=0; i<numofres; i++) if (res[i].sasa >= minsasa && strchr(consensus [0] , res [i] .Itr) ) addchild(-l,i,0) ;

/* (j₀ i consensuslength-1 ' number of child cycles */

for (i=l; i<consensuslength; i++) for (j=numofepires-1; j>=0 && epires [j] .level == i-1; j--)

{ if (strchr (consensus [i] ,'-') ) /* is a gap allowed at this position in the consensus ? */ addchild(j , -l,i) ; if (epires [j] .residue == -1) /* this a gap, so use distance to parents (or older anchestor) instead */ {

/* the following line is for handling multiple gaps after each other */ for (nogapanchestor = epires [j ] .parent; epires [nogapanchestor] .residue == - 1; nogapanchestor = epires [nogapanchestor] .parent) ; for(k=0; k<numofres,- k++) /* if (res[k].sasa >= minsasa && strchr (consensus [i] , res [k] .Itr) && k ! = epires [epires [j] .parent] .residue && sqdist (k, epires [epires [j] .parent] .residue) <= sqmindist) */ if (res[k].sasa >= minsasa && strchr (consensus [i] , res [k] . Itr) && k != epires [nogapanchestor] .residue && sqdist (k, epires [nogapanchestor] .residue) <= sqmindist) addchild(j , k, i) ;

} else

{ for(k=0; k<numofres; k++) if (res [k] .sasa >= minsasa && strchr (consensus [i] , res [k] .Itr) && k != epires [j] .residue && sqdist (k, epires [j] .residue) <= sqmindist) addchild(j , k, i) ; } } longestepitope = epires [numofepires-1] .level+1; }

int cm (const void *a, const void *b)

{ struct epitope *aa = (struct epitope *)a; struct epitope *bb = (struct epitope *)b;

if (aa->sasa < bb->sasa) return 1 ; else if (aa->sasa == bb->sasa) return 0 ; else return -1; }

void processepitopes (void) /* Go through the epitopes, remove copies, nonsense sequences etc. */ { int i, j, k, 1, n, thisepinumbers [MAXCONSENSUS] , processed=0; char thisepi [255] , tmp [50]; char discarded, toobig, onepresent, allpresent; float maxsqdist;

for (i=numofepires-1; i>=0 && epires [i] .level == epires [numofepires-1] .level; i-

-)

{ discarded = 0; toobig = 0; strcpy (thisepi, " ") ; j = i; n = 0; maxsqdist = 0; do { thisepinumbers [n++] = epires [j] .residue; if (epires [j] .residue == -1) /* its a gap */ sprintf (tmp, " , " ) ^■ else sprintf (tmp, "%c%d, ", res [epires [j] .residue] .Itr, res [epires [j ] .residue] .number) ; if (strstr (thisepi, tmp) && epires [j] .residue != -! ) /* only gaps can be present twice! */ discarded = 1; else strcat (thisepi, tmp) ,- j=epires [j] .parent ;

} while (j != -1) ; for (k=0; k <= epires [numofepires-1] . level; k++) for (l=k+l; 1 <= epires [numofepires-1] . level; 1++) if (thisepinumbers [k] != -1 && thisepinumbers [1] != -1) /* if there are no gaps involved */ maxsqdist = max (maxsqdist , sqdist (thisepinumbers [k] , thisepinumbers [1] ) ⁾; if (maxsqdist > sqmaxsize) toobig = 1;

if (toobig) discarded = 1;

if (Idiscarded) /* put the found epitopes into the epitope list */

{ sprintf (epi [numofepitopes] .epi, "%s\n" , thisepi) ; epi [numofepitopes] .sasa = 0; epi [numofepitopes] .gaps = 0; epi [numofepitopes] .residues = 0; epi [numofepitopes] . size = sqrt (maxsqdist) ; for (j = 0; j < n; j++) /* loop over the residues in this epitope */

{ epi [numofepitopes] .res [j] = thisepinumbers [j] ; /* copy the residue numbers to the epitope list */ if (thisepinumbers [j] != -1) /* if it is not a gap */

{ epi [numofepitopes] . sasa += res [thisepinumbers [j] ]. sasa; epi [numofepitopes] .residues++;

} else epi [numofepitopes] .gaps++;

} numofepitopes++ ; if (numofepitopes == MAXEPITOPES)

{ printf ( "MEXEPITOPES exceeded. Increase and recompile program. \n" ) ; exit (0) ;

} }

}

/* now indetify epitopes which are a subset of others */ for (i=0; i<numofepitopes; i++) /* initialize array */ epi [i] . subset = 0 ; for (i=0; i<numofepitopes,- i++) { for (j=0; j<numofepitopes; j++)

{ if (epi [i] .residues > epi [j] .residues)

{ allpresent = 0; for (k=0; k<epi [i] . residues; k++)

{ if (epi [i] .res [k] != -1)

{ onepresent = 0; for (1=0; l<epi [j] .residues; 1++) if (epi [i] .res [k] == epi [j] .res [1] ) /* if the residues are the same and not gaps */ onepresent = 1; allpresent |= onepresent;

} } if (allpresent)

{ epi [j] .subset = 1;

/* numofsubsets++; */

} } } }

/* now sort the epitopes according to SASA */ qsort (& (epi [0] ), numofepitopes, sizeof (struct epitope), &cmp) ,-

/* counts the ones that are subsets of others */ for (i=0; i<numofepitopes; i++) if (epi [i] .subset == 1) numofsubsets++; /* now count how many epitopes each ressidue is a member of, considering only non-redundant epitopes, and the number of epitopes wanted */ for (i=0; i < numofepitopes && processed < maxepi; i++) if (epi [i] .subset == 0) /* count only if the epitope is not a subset of another */

{ processed++; for (j=0; j < epi [i] .residues,- j++)

(res [epi [i] .res [j] ] .member_of_epitopes) ++; /* add the counter for epitopes for the residues */

} }

void printepitopes (void)

{ int i, processed = 0; for (i=0; i < numofepitopes && processed < maxepi; i++) if (epi [i] .subset == 0)

{ printf ("SAS: %3d, Size %5.2f: %s" , epi [i] . sasa, epi [i]. size, epi [i] . epi) ; processed++;

}

void usage (void)

{ fprintf (stderr, "USAGE: epitope <epitope template> <filename_template> dist ace maxsize number minlength\n" ) ,- fprintf (stderr, "\n") ,• fprintf (stderr, "filenames <filename_template> .pdb and <file- name_template>.dssp\n") ; fprintf (stderr, " must be present . \n") ; fprintf (stderr, "dist is the maximum distance between adjacent residues in epi- tope.\n"); fprintf (stderr, "ace is minimum surface accessible area in square angstroms. \n" ) ; fprintf (stderr, "maxsize is the maximum distance between any two residues in the epitope . \n" ) ; fprintf (stderr, "number is the maximum number of non-redundant epitopes to consider (0=all)\n")_; fprintf (stderr, "minlength is the minimum length of the epitope seqs (in fractions^") ,- fprintf (stderr, " of the consensus sequence length) . \n" ) ; fprintf (stderr, "A file <filename_template> .dat containing the number of epi- topes\n") ; fprintf (stderr, "each residue participates in is written. \n" ) ; fprintf (stderr, "\n") ,- exit (0) ; }

int main (int argc, char **arg) { int i; char pdbf ile [256] , dsspf ile [256] , datf ile [256] ; if (argc ! = 8 ) usage ( ) ; readconsensus (arg [1] ) ; printf ( "Epitope consensus sequence read from %s\n" , arg [1] ) ; printf ( " \n" ) ; for (i = 0; i < consensuslength; i++) printf ("%s\n" , consensus [i] ) ; printf ("\n") ; strcpy(pdbfile, arg [2] ) ,- strcat (pdbfile, " .pdb") ; strcpy(dsspfile, arg [2] ) ,- strcat (dsspfile, " .dssp") ,- strcpy (datfile, arg [2] ) ; strcat (datfile , " . dat " ) ; readpdbCA (pdbfile) ; printf ("Sequence read from %s\n" , pdbfile) ; printf (" \n") ; for (i = 0; i < numofres; i++)

{ printf ("%c", res [i] .Itr) ; if (I ((i+l)%70)) printf ("\n") ; } printf ("\n\n") ,- readdssp (dsspfile) ; mindist = atof (arg [3]) minsasa = atoi (arg [4]) maxsize = atof (arg [5]) maxepi = atoi (arg [6]) if (maxepi == 0) maxepi = INT_MAX; minlength = atof (arg [7] ) ,- /* minimum length of epitope sequence (in fractions of the consensus length) */ sqmindist = mindist*mindist ; sqmaxsize = maxsize*maxsize; minlength_residues = (float) ceil (minlength*consensuslength) ,- findepitopes () ; if (longestepitope >= minlength_residues) processepitopes () ; printf "Parameters and internal numbers\n"); printf .. \_n..) . printf "Program revision date %s\n", REVISIONDATE) ; printf "Consensus sequence length %d\n" , consensuslength) ; printf "Minimum epitope seq length threshold %.2f (%d residues) \n" , minlength, minlength_residues) ; printf "Longest epitope sequence found %d\n" , longestepitope) ; printf "Number of residues in PDB file %d\n", numofres) ; printf "Distance threshold value (angstroms) %.lf\n " , mindist) ; printf "Minimum surface accessible area of each res %d\n" , minsasa) ; printf "Maximum epitope size %.lf\n " , maxsize) ; printf "Number of nodes in epitope tree %d\n", numofepires) ; printf "Total number of epitopes.... %d\n" , numofepitopes) ; printf "....of which are subsets of others %d\n" , numofsubsets) ; printf "Max number of non-redundant epitopes %d\n" , maxepi) ; printf "\n")_; printf ("Epitopes found\n"); printf (" \n")_; if (longestepitope >= minlength_residues) printepitopes ( ) ; writedataf ile (datf ile) ,-

/* for (i = 0; i < numofepires; i++) printf ("|%4d %4d %4d %4d ",i, epires [i] .level, epires [i] .residue, epires [i] .parent) ;

*/ return 0 ;

}

Appendix B The wrapper (Python) (e i o es . cgi)

# ! /z/vaks/bin/python

#

# Automatic epitope mapping

# import cgi, os, time, commands, string, sys

FormFile = "epitope.html" scriptdir = "/z/edhome/eρf/public_html/epitope/" epitopepath = "/z/edhome/epf/epitope/epitope3" dssppath = "/z/vaks/bin/dssp" gnuplotpath = "/z/edhome/epf/gnuplot-3.7/gnuplot" zippath = "/usr/freeware/bin/zip" unzippath = "/usr/freeware/bin/unzip" timestamp = str (int (time. time ()) ) liball = range (1,53) libigg = [3,4,7,11,14,16,17,30,31,32,34,35,38,39,41,42,43,47,48,49,50,51,52] libige =

[1,2,5,6,8,9,10,12,13,15,18,19,20,21,22,23,24,25,26,27,28,29,33,36,37,40,44,45,46]

# the page startes here print "Content-type: text/html\n\n" # HTML is following print ^,<html>\n^l print '<head>\n' print ' <title>Automatic epitope mapping</title>\n' print '</head>\n^τ print ' \n'

# check for lock file if os.path.isfile ("epitope. lock") : print 'Sorry - lock file exists. This means that automatic epitope mapping is al- ready in use, ' print 'or that an error has occured.<BR> ' print "If you are absolutely sure that no one are using automatic epitope mapping, you can" print "press the button below. <BR>" print "If you are not sure, just press 'back' in your browser now." print ' <BRxBR> ' print ' <form METH0D=GET AC- TION="http : //vaks .novo. dk/~epf/epitope/epitope_removelock. cgi" ><input tyρe="submit" name="SUBMIT_BUTTON" value="Remove lock file"x/form> ' sys. exit (0)

# create lock file os. system ("touch epitope. lock")

# Clean up directory

# (delete everything but md_analysis.cgi and md_analysis.html) #commands. getoutput ("Is -1 | awk '$9 !- /^Λepitope/ {print \"rm\",$9}' >cleanup. sh") ttcommands .getoutput ( " . "+scriptdir+ "cleanup . sh" )

#if os .path. isfile ("cleanup. sh") :

# os . remove ( "cleanu . sh" ) commands .getoutput ("rm *.png") commands .getoutput ( "rm * . da . txt" ) commands .getoutput ("rm *. out. txt")

# remove any subdirs commands. getoutput ("find . -type d -name '???*' -exec rm -rf {} \;")

# the page continues here form = cgi . FieldStorage ( )

infile = form ["pdbfile"] .value namebase = form["pdbfile"] .filename namebasenum = string .rfind(namebase, ' \\ ' ) if namebasenum < -1: namebasenum = 0 namelist = string . split (namebase [namebasenum+1 :],'.') pdbname = namelist [0] + ' .pdb' dsspname = namelist [0] +' .dssp ' datname = namelist [0] +'. dat ' dattxtname = namelis [0] +' .dat .txt ' zipname = namelist [0] +'. zip¹ inzipname = 'submitted.zip' consensusname = namelist [0] +'. cons ' epiname = namelist [0] +' .out . txt ' minsasa = form ["minsasa"] .value mindist = form ["mindist"] .value maxsize = for ["maxsize"] .value consensus = form ["consensus"] .value threshold = form ["threshold"] .value number = for ["number"] .value minlength = form ["minlength"] .value plotmode = form["plot_mode"] .value operatemode = form["operate_mode"] .value if (operatemode [0 : 7] == "library"): operatemode = "library" if (form ["operate_mode"] .value == "library_all") : lib = liball elif (form ["operate_mode"] .value == "library_igg") : lib = libigg elif (form["operate_mode"] .value == "1ibrary_ige" ) : lib = libige if (operatemode == "library"): libsize = len(lib) if (string.upper (namelis [1] ) == 'PDB'): inputtype = 'PDB' if (string.upper (namelist [1] ) == 'ZIP'): inputtype = 'ZIP'

# write submitted file if (inputtype == 'PDB'): f=open(pdbname, "w") if (inputtype == 'ZIP'): f=open(inzipname, "w") f .write (infile) f .close ()

# If the submitted file is a zip-file, extract it and make a list of the en- tries if (inputtype == 'ZIP'): pdbfiles = string. split (commands.getoutput (unzippath+" -1 "+inzipname+" | awk '{ if (NR > 3 && NF == 4) print $4}'")) numofpdbfiles = len (pdbfiles) commands .getoutput (unzippath+" -j "+inzipname)

# make directories and move the zipfiles there for i in pdbfiles: dirname = i [0 : -4] commands .getoutput ("rm -rf "+dirname) os .mkdir (dirname) os. rename (i,dirname+"/"+i) else: pdbfiles = [pdbname]

# if (operatemode == "single"): f=open(consensusname, "w") f .write (consensus) f. closet)

print '<CENTER>\n' if form.has_key ("pagetitle") : print '<Hl>'+form[ "pagetitle"] .value+ ' </Hl>\n' print time .ctime (time . time 0 ) + ' <BR><BR>\n' if (operatemode == "single") : print '<BRxH2>You should print or save this page! </H2>\n' print 'The results shown on this page are not stored anywhere else.\n\n' if (operatemode == "library") : if (inputtype == 'ZIP'): print '<H2xA HREF="collected. zip">Download</A> your results ! </H2>\n' if (inputtype == 'PDB'): print '<H2xA HREF=" ' +zipname+ ' ">Download</A> your results ! </H2>\n' print 'Downloading is strongly recommended! The results are shown on this page and included\n' print 'in this archive. They are not stored anywhere else . <BRxBR>\n' print 'Filename given by you:<BR>\n' print '<B> ' +form[ "pdbfile"] . filename+ ' </B>\n'

run the program

#if (inputtype == 'ZIP'): if (1 == 1) : for currentpdbname in pdbfiles: # the naming stuff - identical to that at the top of the file namebase = currentpdbname namebasenum = string. rfind (namebase, ' \\ ' ) if namebasenum < -1: namebasenum = 0 namelist = string . split (namebase [namebasenum+1 :],'.') if (inputtype == 'PDB'): nameroot = namelist [0] if (inputtype == 'ZIP'): nameroot = namelist [0] # nameroot = currentpdbname [0 : -4] +"/"+namelist [0] pdbname = nameroot+ ' .pdb' dsspname = nameroot+ ' .dssp' datname = nameroot+ ' . da ' dattxtname = nameroot+ ' . dat . txt ' zipname = nameroot+' .zip' epiname = nameroot+ ' . out . xt '

# here comes the treatment of the individual structures if (inputtype == 'ZIP'): os . chdir (currentpdbname [0 : -4] ) if (operatemode == "single") :

# add extra newlines to the consensus file commands . getoutput ("echo \\\\n\\\\n >> "+consensusname) commands.getoutput (dssppath+" "+pdbname+" "+dsspname) if (inputtype == 'ZIP'): commands .getoutput (epitopepath+" .. /"+consensusname+" "+namelist [0] +" "+mindist+" "+minsasa+" "+maxsize+" "+number+" "+minlength+" > "+epiname) else: commands .getoutput (epitopepath+" "+consensusname+" "÷namelis [0] +" "+mindist+" "+minsasa+" "+maxsize+" "+number+" "+minlength+" > "+epiname) commands .getoutput ( "mv "+datname+" "+dattxtname) if (operatemode == "library"): commands.getoutput (dssppath+" "+pdbname+" "+dsspname) # for i in range (1, libsize+1) : for i in lib: if (inputtype == 'ZIP'): commands. getoutput (epitopepath+" ../"+string. zfill(str(i),3)+" .epi "+namelist [0] +" "+mindist+" "+minsasa+" "+maxsize+" "+number+" "+minlength+ " > "+string.zfill(str(i) ,3)+" .out. txt") else: commands . getoutpu (epitopeρath+" "+string.zfill (str (i) ,3) +" .epi "+namelist [0] +" "+raindist+" "+minsasa+" "+maxsize+" "+number+" "+minlength+" > "+string.zfill(str(i) ,3)+" .out. txt") commands. getoutput ("mv "+datname+" "+string. zfill (str (i) ,3) +" .dat .txt") residues = in (commands.getoutput ("grep -v '#' "+string.zfill(str(lib[0] ) ,3) +" .dat .txt | wc | awk '{print $l}'")) commands . getoutpu ( "rm sum . dat . txt " ) for i in range (1, residues+1) : grepstr = "^Λ"+string.rjust (str (i) ,4) commands.getoutput ("grep ' "+grepstr+" ' *. dat. txt | awk 'BEGIN{sum=θ} {sum+=$5; res=$2; pdbres=$3; AA=$4} END{print res, pdbres, AA,sum}' >> sum.dat.txt") commands .getoutput { "rm "+datname) collect generated files if (inputtype == 'PDB'): commands .getoutput ("rm "+zipname) commands .getoutput (zippath+" "+zipname+" *.out. txt *. dat. txt")

if in library mode, create and show the sum graph

if (operatemode == "library") : timestamp = str (int (time. time() ) ) f=open("epitope.gnp" , "w") if (plotmode == "sequential"): f.writet'set xlabel "Residue number (sequential) "\n' ) else: f.writet'set xlabel "Residue number (PDB)"\n') f.write('set ylabel "Epitopes"\n' ) f.writepset title " ' +currentpdbname [0 : -4] + ' "\n' ) f.write('set size ratio 0.3 1, 0.5\n') f .write ('set term png small color\n') f.writet'set out "epi ' +timestamp+ ' .png"\n' ) if (plotmode == "sequential"): f .write ( 'plot "sum.dat.txt" using 1:4 title "Number of epitopes" with steps 1, '+threshold+' title "Threshold" with lines 3\n') else-. f .write ( 'plot "sum.dat.txt" using 2:4 title "Number of epitopes" with steps 1, ' +threshold+ ' title "Threshold" with lines 3\n') f.closeO commands .getoutpu (gnuplotpath+" epitope .gnp" ) print '<Hl>Epitope frequency sums for each residue</HlxBR>\n' if (form["operate_mode"] .value == "library_all") : print '<H2>Library of ' +str (libsize) +' epitopes (IgG+IgE) </H2> ' elif (form["operate_mode"] .value == "1ibrary_igg" ) : print '<H2>Library of ' +str (libsize) + ' epitopes (IgG)</H2>' elif (form["operate_mode"] .value == "library_ige") : print '<H2>Library of ' +str (libsize) + ' epitopes (IgE)</H2>' if (inputtype == 'PDB'): print '<BRxBRxIMG SRC="epi ' +timestamp+ ' .png"xBR><BR>\n' print ' <A HREF= "sum. dat . txt">View the frequency sums table data</AxBR>\n' print '<A HREF=" ' +zipname+ ' ">Download</A> a zip file with all results from the individual epitopes . <BR>\n' print '</CENTER>\n' if (inputtype == 'ZIP'): print '<BRxBRxIMG SRC=" ' +currentpdbname [0 : - 4] + ' /epi '+timestamp+' .png"xBR><BR>\n' print ' <A HREF=" '+currentpdbname [0 : -4] +' /sum.dat .txt" >View the frequency sums table data</A><BR>\n'

# now make gnuplot graphs and data lists for individual epitopes

# so far this goes only for the "single" operating mode if (operatemode == "single"): timestamp = str (int (time. ime ()) )

# Create gnuplot control file f=open( "epitope .gnp", "w") if (plotmode == "sequential"): f. write ('set xlabel "Residue number (sequential) "\n' ) else: f. rite ('set xlabel "Residue number (PDB)"\n') f.write('set ylabel "Epitopes"\n' ) f. write {'set size ratio 0.3 1, 0.5\n') f. write ('set term png small color\n') f. write ('set out "epi ' +timestamp+ ' .png"\n' ) if (plotmode == "sequential"): f .write ( 'plot " ' +dattxtname+ ' " using 1:4 title "Number of epitopes" with steps 1, ' +threshold+ ' title "Threshold" with lines 3\n') else: f .write ( 'plot " ' +dattxtname+ ' " using 2:4 title "Number of epitopes" with steps 1, '+threshold+' title "Threshold" with lines 3\n') f .close () commands .getoutpu (gnuplotpath+" epitope .gnp" ) if (inputtype == 'ZIP'): print ' <BRxBRxIMG SRC=" ' +currentpdbname [0 : - 4] + ' /epi '+timestamp+ ' .png"xBR><BR>\n' print ' <A HREF=" '+currentpdbname [0 : -4] + '/ ' +dattxtname+ ' ">View the table da- ta</AxBR>\n' else: print '<BRxBRxIMG SRC="epi ' +timestamp+ ' .png"xBRxBR>\n' print ' <A HREF=" ' +dattxtname+ ' ">View the table data</AxBR>\n' print '</CENTER?\n'

print the table print ' <PRE> ' f=open(epiname, "r") line = f.readlineO while line != "" -. line = string. replace (line, ' \n' ,'' ) print line line = f.readlineO f .close () print '</PRExBRxBRxBR>'

if (inputtype == 'ZIP') os . chdir ("..")

# f_or zip-mode (library only) : count number of epitopes found from each lib consensus

if (inputtype == 'ZIP' and operatemode == "library"): numofepitopes = [] f=open ( "epitopecoun . txt" , "w" ) f .write (string.1just ("PDB file", 20) ) for i in lib: f .write (string. rjust (str (i) , 6) ) f .write C\n' ) for j in range (len (pdbfiles) ) : currentpdbname = pdbfiles [j] f . rite (string.1just (currentpdbname [0 :20] ,20) ) for idx in range (len (lib) ) : i = lib [idx] filename = currentpdbname [0 : -4] +"/"+string. zfill (st (i) , 3) +" .out . txt" numofepitopes . append (0) tmp = commands.getoutput ("grep 'Total number of epitopes' "+filename+" | awk ' {print $6} ' ") if (tmp != "") : numofepitopes [j*len (pdbfiles) +idx] = int (tmp) numofepitopes [j*len (pdbfiles) +idx] = numo epitopes [j *le (pdbfiles) +idx] - int (commands .getoutput ("grep 'of which are subsets' "+filename+" | awk '{print $8}'")) else: numofepitopes [j*len (pdbfiles) +idx] = 0 f .write (string. rjust (st (numofepitopes [j*len (pdbfiles) +idx] ) , 6) ) f .write ( '\n') f .close() # for ZIP-mode: Collect all dirs and files if (inputtype == 'ZIP'): commands .getoutput ( "rm collected. zip" ) for currentpdbname in pdbfiles: commands .getoutput (zippath+" -r -u collected.zip "+currentpdbname [0 : -4] ^' if (operatemode == "library"): commands .getoutput (zippath+" -u collected.zip epitopecount.txt") # Last lines

print '</body>\n' print '</html>\n'

# remove lock file os. remove ("epitope. lock") # remove temporary files

#if (inputtype == 'ZIP'):

# for currentpdbname in pdbfiles :

# commands .getoutput ( "rm -rf "+currentpdbname [0 : -4] ) commands .getoutput ("rm "+pdbname) commands . getoutput ("rm "+dsspname) commands .getoutput ("rm "+consensusname) commands. getoutput ("rm "+epiname) Appendix C The HTML input form (epi topes. html)

<!doctype html public "-//w3c//dtd html 4.0 transitional//en"> <html> <head> <meta http-equiv="Content-Type" content=" text/html; charset=iso-8859-l">

<title>Automatic epitope mapping</title> </head> <B0DY BGC0L0R="#FFF9E6" text="#000000 " link="#000040 " vlink="#404040"> <center>

< TABLE >

<TR>

<TD> &nbsp,- &nbsp,-S:nbsp;<Hl>Epitope mapping tool </Hlx/TD> </TR>

</ TABLE >

</center> <form ENCTYPE= "multipart/form-data" action=" ./epitope5.cgi" method="POST">

<H2>Title</H2>

Page title:   <INPUT type=text name="pagetitle" size="40" maxlength="80" value="Automatic Epitope Mapping">

<H2>Parameters</H2>

<TABLE>

<TR> <TD>File name (on your local machine) </TD>

</TR>

<TRxTD COLSPAN=2>You may submit either a PDB file containing a single structure or a ZIP-archive containing a number of PDB files, each defining a single structure. The ZIP-archive must not contain subdirectories.

</TABLE>

&nbsp,- &nbsp,-Use epitope library (Full library) . <BR>

   Use epitope library (IgG library) . <BR>

<INPUT TYPE=RADIO NAME="operate_mode" VALUE="library_ige">    Use epitope library (IgE library) . <BR>

&nbsp,- &Lnbsp;Specify epitope consensus sequence here : <BR>

Epitope consensus sequence<BR>

</TEXTAREA> < ITD_>

</TDxTD> <TD>

Example of consensus sequence input :<BR>

<BR>

<TRxTD>KR </TDxTDx/TDxTD> (Lys og Arg allowed) </TDxTR> <TRxTD>AILV-</TDxTDx/TDxTD> (Ala, Ile, Leu, Val or missing residue allowed) </TDxTR> <TRxTD>* </TDxTDx/TDxTD> (All residues allowed, but there must be a residue) </TDxTR>

<TRxTD>? </TDxTDx/TDxTD> (All or missing residue allowed) </TDxTR> <TRxTD>DE </TDxTDx/TDxTD> (Asp or Glu allowed) </TDxTR> </TABLE> <BR>

*, ? or - in first or last position is allowed but obsolete. (- in first position is ignored.) </TDx/TR> </TABLE> <BRxHR WIDTH=80%xBR>

< ABLE> <TR>

<TD>Maximum distance between adjacent residues </TDxTDxINPUT type=text na- me= "mindist" size="5" maxlength="8" value = "10"></TD>

</TR>

<TR> <TD>Minimum solvent accessible surface area for each residue</TDxTD><INPUT type=text name= "minsasa" size="5" maxlength="8" value = "5"x/TD>

</TR>

<TR>

<TD>Maximum epitope size (max distance between any two residues in epi- tope) </TDxTDxINPUT type=text name= "maxsize" size="5" maxlength="8" value =

"25"x/TD>

</TR>

<TR>

<TD>Maximum number of non-redundant epitopes to include (0 = all) </TDxTDxINPUT type=text name="number" size="5" maxlength="8" value = "0"x/TD>

</TR>

<TD>Minimum epitope sequence length (in fractions of consensus length) </TDxTD^χINPUT type=text name= "minlength" size="5" maxlength="8" value = "0.80"></TD>

</TR> </TABLE>

<H2>Graph</H2>

<INPUT TYPE=RADIO NAME="plot_mode" VALUE=" sequential" CHECKED> &hbsp;  Use sequential numbering of residues . <BR> <INPUT TYPE=RADIO NAME="plot_mode" VALUE="pdb">

  &nbsp,-Use PDB numbering of residues. (Will sometimes produce funny results. ) <BR> Threshold value  &nbsp,-&nbsρ,-<INPUT type=text name="threshold" size="5" max- length="8" value = "2"><BR>

Comments and bug reports to <A HREF= "mailto : epf@novo . dk" >epf </A> .

<BRxBR>

</body>

</html> Appendix D

3D Structure of Esperase

ATOM 1 N GLN A 2 24 .343 43, .495 26 .356 1, .00 26 .00 7

ATOM 2 NE2 GLN A 2 25 .686 39 .582 30 .163 1 .00 20 .88 7

ATOM 3 OE1 GLN A 2 23 .497 39 .261 29 .938 1, .00 23 .07 8

ATOM 4 CD GLN A 2 24 .448 40, .036 29 .883 1, .00 23 .09 6

ATOM 5 CG GLN A 2 24 .420 41 .507 29 .607 1 .00 23 .93 6

ATOM 6 CB GLN A 2 24 .309 41 .801 28 .125 1, .00 23 .06 6

ATOM 7 CA GLN A 2 23 .999 43, .235 27 .778 1, .00 25 .53 6

ATOM 8 C GLN A 2 24 .957 44 .096 28 .566 1 .00 28 .66 6

ATOM 9 O GLN A 2 26. .126 44, .049 28, .148 1, .00 31, .97 8

ATOM 10 N THR A 3 24, .538 44, .857 29, .557 1. .00 25, .20 7

ATOM 11 CG2 THR A 3 24 .948 47, .593 29 .045 1, .00 32 .60 6

ATOM 12 OG1 THR A 3 23 .634 46, .905 30 .890 1. .00 33, .76 8

ATOM 13 CB THR A 3 24 .979 47, .085 30 .464 1, .00 26, .52 6

ATOM 14 CA THR A 3 25 .508 45, .643 30 .316 1, .00 24 .44 6

ATOM 15 C THR A 3 25 .551 45, .035 31 .717 1, .00 23, .97 6

ATOM 16 O THR A 3 24 .566 44 .377 32 .092 1, .00 27 .28 8

ATOM 17 N VAL A 4 26 .585 45, .366 32 .449 1, .00 24 .67 7

ATOM 18 CG2 VAL A 4 28 .377 43, .274 33 .058 1, .00 22 .99 6

ATOM 19 CGI VAL A 4 28 .147 43, .784 35, .492 1. .00 22, .90 6

ATOM 20 CB VAL A 4 28 .128 44 .351 34 .069 1, .00 24 .23 6

ATOM 21 CA VAL A 4 26 .694 44, .897 33 .837 1, .00 24 .05 6

ATOM 22 C VAL A 4 26 .445 46 .114 34 .776 1, .00 22 .35 6

ATOM 23 O VAL A 4 27, .323 47. .015 34, .816 1. .00 24, .67 8

ATOM 24 N PRO A 5 25, .365 46. .082 35, .507 1. .00 21, .36 7

ATOM 25 CD PRO A 5 24 .284 45, .039 35, .492 1. .00 16, .33 6

ATOM 26 CG PRO A 5 23, .100 45, .761 36, .119 1. .00 19, .38 6

ATOM 27 CB PRO A 5 23, .741 46. .724 37, .115 1. .00 17, .69 6

ATOM 28 CA PRO A 5 25, .049 47. .159 36, .454 1. ,00 17, .81 6

ATOM 29 C PRO A 5 26, .231 47, .367 37, .382 1, .00 24, .17 6

ATOM 30 O PRO A 5 26, .903 46, .375 37, .763 1. .00 19, .11 8

ATOM 31 N TRP A 6 26 .505 48, .602 37, .832 1. .00 21, .75 7

ATOM 32 CD2 TRP A 6 26, .928 50, .889 41, .509 1. .00 18, .89 6

ATOM 33 CE3 TRP A 6 27. .995 50, .522 42, .349 1. ,00 19, .68 6

ATOM 34 CZ3 TRP A 6 27 .789 50, .639 43, .721 1. .00 18, .65 6

ATOM 35 CH2 TRP A 6 26 .582 51, .111 44, .306 1. .00 18, .90 6

ATOM 36 CZ2 TRP A 6 25, .524 51, .469 43, .465 1. .00 18, .51 6

ATOM 37 CE2 TRP A 6 25, .705 51. .348 42, .088 1. ,00 24. .32 6

ATOM 38 NΞ1 TRP A 6 24, .852 51. ,593 41. .020 1. ,00 22. ,59 7

ATOM 39 CD1 TRP A 6 25, .420 51. ,300 39. .828 1. 00 14. ,24 6

ATOM 40 CG TRP A 6 26, .698 50, .865 40. .074 1. ,00 17, .07 6

ATOM 41 CB TRP A 6 27, .702 50. .382 39. .095 1. ,00 19. .96 6

ATOM 42 CA TRP A 6 27, .668 48, .899 38. .677 1. ,00 19. .10 6

ATOM 43 C TRP A 6 27, .699 48, .015 39, .926 1. ,00 20, .24 6

ATOM 44 O TRP A 6 28, .865 47. .719 40. .230 1. ,00 19. .68 8

ATOM 45 N GLY A 7 26. .553 47. ,779 40. .554 1. ,00 19. .54 7

ATOM 46 CA GLY A 7 26, .573 47. .016 41, .827 1. ,00 15, .44 6

ATOM 47 C GLY A 7 27, .075 45. .596 41. .634 1. ,00 21. .44 6

ATOM 48 O GLY A 7 27, .733 45. ,067 42. .534 1. .00 20. .88 8

ATOM 49 N ILE A 8 26, .862 44, .983 40, .482 1. ,00 19, .17 7

ATOM 50 CD1 ILE A 8 24, .548 42, .180 39, .852 1. ,00 19, .08 6

ATOM 51 CGI ILE A 8 25, .219 43, .020 38, .790 1. ,00 17, .53 6

ATOM 52 CB ILE A 8 26, .746 43. .093 38, .871 1. ,00 23, .00 6

ATOM 53 CG2 ILE A 8 27, .338 41. .799 38. .350 1. ,00 22. .68 6

ATOM 54 CA ILE A 8 27, .325 43, .598 40, .192 1. ,00 23, .07 6

ATOM 55 C ILE A 8 28, .853 43. .585 40, .232 1. ,00 22, .71 6

ATOM 56 O ILE A 8 29, .462 42. .674 40. .821 1. ,00 21. .85 8

ATOM 57 N SER A 9 29, .527 44. .534 39. .631 1. ,00 19. .30 7 ATOM 58 OG SER A 9 31,.089 45,.298 37,.438 1..00 28..25 8

ATOM 59 CB SER A 9 31. .514 45 .590 38, .718 1, .00 24, .45 6

ATOM 60 CA SER A 9 30, .986 44, .532 39, .663 1, .00 18, .00 6

ATOM 61 C SER A 9 31, .431 45 .071 41, .000 1, .00 18, .16 6 ATOM 62 O SER A 9 32, .543 44 .676 41, .351 1, .00 21, .78 8

ATOM 63 N PHE A 10 30, .702 45, .961 41, .617 1, .00 17. .83 7

ATOM 64 CD2 PHE A 10 31 .780 49 .344 44, .181 1, .00 23, .83 6

ATOM 65 CE2 PHE A 10 32, .100 50, .259 45, .170 1, .00 27, .32 6

ATOM 66 CZ PHE A 10 31, .514 50 .266 46, .431 1, .00 21, .18 6 ATOM 61 CE1 PHE A 10 30, .563 49 .309 46, .768 1, .00 29, .76 6

ATOM 68 CD1 PHE A 10 30 .188 48 .429 45, .759 1 .00 23 .23 6

ATOM 69 CG PHE A 10 30, .778 48 .438 44, .521 1, .00 18, .74 6

ATOM 70 CB PHE A 10 30. .285 47. .522 43. ,455 1. ,00 17. ,70 6

ATOM 71 CA PHE A 10 31. .270 46, .528 42. .864 1, .00 20. .00 6 ATOM 72 C PHE A 10 31. .457 45, .396 43. .870 1. .00 22. .92 6

ATOM 73 O PHE A 10 32, .357 45, .569 44, .723 1, .00 24, .39 8

ATOM 74 N ILE A 11 30. .614 44, .376 43. .829 1. .00 19. .21 7

ATOM 75 CD1 ILE A 11 27, .476 41 .276 44, .648 1, .00 14, .26 6

ATOM 76 CGI ILE A 11 28. .743 41, .954 44. .149 1, .00 18, .25 6 ATOM 77 CB ILE A 11 29, .500 42 .669 45, .229 1, .00 23, .27 6

ATOM 78 CG2 ILE A 11 28, .762 43, .839 45. .866 1, .00 21, .09 6

ATOM 79 CA ILE A 11 30, .789 43, .259 44. .739 1, .00 20. .52 6

ATOM 80 C ILE A 11 31. .715 42 .170 44, .172 1, .00 21, .46 6

ATOM 81 O ILE A 11 31, .783 41, .155 44. .840 1, .00 20, .99 8 ATOM 82 N ASN A 12 32, .378 4 .329 43, .056 1, .00 21, .03 7

ATOM 83 ND2 ASN A 12 35, .345 43. .095 44, .578 1, .00 30, .69 7

ATOM 84 OD1 ASN A 12 36. .135 42, .268 42. .569 1. .00 35. .13 8

ATOM 85 CG ASN A 12 35, .390 42 .276 43, .541 1, .00 25, .00 6

ATOM 86 CB ASN A 12 34. .450 41, .092 43. .449 1. .00 21. .03 6 ATOM 87 CA ASN A 12 33. .340 41, .412 42. .463 1. .00 23. .98 6

ATOM 88 C ASN A 12 32. .735 40, .088 41. ,978 1. .00 24. ,79 6

ATOM 89 O ASN A 12 33. .438 39, .085 42. .118 1. .00 23. .07 8

ATOM 90 N THR A 13 31, .520 40, .204 41, .505 1, .00 20, .38 7

ATOM 91 CG2 THR A 13 28. .654 38, .417 39. .642 1. .00 15, .01 6 ATOM 92 OG1 THR A 13 28, .704 40 .013 41, .326 1, .00 22, .51 8

ATOM 93 CB THR A 13 29, .488 39, .474 40, .308 1, .00 19, .67 6

ATOM 94 CA THR A 13 30, .810 39 .083 40, .956 1, .00 20, .28 6

ATOM 95 C THR A 13 31, .671 38 .384 39, .892 1, .00 21, .19 6

ATOM 96 O THR A 13 31. .605 37, .158 39, .791 1, .00 23, .59 8 ATOM 97 N GLN A 14 32, .334 39 .049 39, .028 1, .00 20, .22 7

ATOM 98 NE2 GLN A 14 32. .431 41, .889 38. ,600 1. .00 33. ,33 7

ATOM 99 OE1 GLN A 14 31. .706 42, .497 36. .548 1. .00 50. .01 8

ATOM 100 CD GLN A 14 32. .245 41, .660 37. .297 1. .00 52. ,65 6

ATOM 101 CG GLN A 14 32. .764 40, .430 36. .555 1, .00 52, .84 6 ATOM 102 CB GLN A 14 33. .857 39, .542 37. .128 1. .00 28. .62 6

ATOM 103 CA GLN A 14 33. .138 38, .429 37. .955 1. .00 32. ,46 6

ATOM 104 C GLN A 14 34. .201 37, .476 38. .497 1, .00 31. .89 6

ATOM 105 O GLN A 14 34. .509 36, .571 37. .705 1. .00 27. .29 8

ATOM 106 N GLN A 15 34, .744 37 .757 39, .679 1, .00 23, .92 7 ATOM 107 NE2 GLN A 15 38, .511 39, .924 42. .603 1. .00 44. .05 7

ATOM 108 OE1 GLN A 15 37, .542 38 .314 43, .749 1, .00 38, .30 8

ATOM 109 CD GLN A 15 37, .762 38 .831 42. .664 1, .00 40, .79 6

ATOM 110 CG GLN A 15 37 .188 38 .390 41, .331 1, .00 34, .24 6

ATOM 111 CB GLN A 15 36, .297 37 .200 41, .508 1, .00 24, .39 6 ATOM 112 CA GLN A 15 35. .728 36, .783 40. .170 1. .00 22. .62 6

ATOM 113 C GLN A 15 35, .042 35 .443 40. .384 1, .00 29, .48 6

ATOM 114 O GLN A 15 35, .749 34, .432 40. .285 1. .00 31. .32 8

ATOM 115 N ALA A 16 33, .762 35 .385 40, ,769 1, .00 23, .78 7

ATOM 116 CB ALA A 16 31, .804 34 .146 41, .761 1, .00 18, .00 6 ATOM 117 CA ALA A 16 33, .069 34 .097 40, .925 1, .00 21, .90 6

ATOM 118 C ALA A 16 32 .825 33 .561 39 .502 1, .00 26. .74 6 ATOM 119 O ALA A 16 32.967 32,.352 39.191 1,.00 30,.41 8

ATOM 120 N HIS A 17 32 .281 34 .385 38 .577 1 .00 30 .64 7

ATOM 121 CD2 HIS A 17 29 .257 34 .877 38 .233 1 .00 22 .07 6

ATOM 122 NE2 HIS A 17 28 .016 35 .453 38 .259 1, .00 25, .33 7 ATOM 123 CE1 HIS A 17 27, .909 36, .328 37 .220 1, .00 20, .45 6

ATOM 124 ND1 HIS A 17 29, .020 36. .372 36, .515 1, .00 24, .91 7

ATOM 125 CG HIS A 17 29 .849 35, .428 37 .109 1, .00 22, .09 6

ATOM 126 CB HIS A 17 31, .222 35, .150 36 .543 1, .00 19, .27 6

ATOM 127 CA HIS A 17 31, .865 33, .972 37 .219 1, .00 19, .98 6 ATOM 128 C HIS A 17 33 .073 33, .367 36 .512 1 .00 29, .30 6

ATOM 129 O HIS A 17 32 .959 32, .347 35 .823 1, .00 27, .69 8

ATOM 130 N ASN A 18 34 .191 34 .028 36 .705 1 .00 28 .18 7

ATOM 131 ND2 ASN A 18 36, .859 36, .788 35, .613 1, .00 45, .93 7

ATOM 132 OD1 ASN A 18 35 .325 35, .559 3 .498 1, .00 40, .29 8 ATOM 133 CG ASN A 18 36 .220 35, .663 35 .347 1, .00 40, ,01 6

ATOM 134 CB ASN A 18 36, .641 34, .520 36 .270 1, .00 30, .63 6

ATOM 135 CA ASN A 18 35 .432 33, .605 36 .085 1, .00 27, .13 6

ATOM 136 C ASN A 18 35 .838 32, .250 36 .577 1, .00 35, .11 6

ATOM 137 O ASN A 18 36, .705 31, .803 35 .846 1, .00 35, .07 8 ATOM 138 N ARG A 19 35 .399 31, .756 37 .675 1 .00 32 .73 7

ATOM 139 NH2 ARG A 19 35, .515 32. .617 44, .021 1. .00 53, .72 7

ATOM 140 NH1 ARG A 19 37. .640 32. .800 43, .686 1. .00 51. .43 7

ATOM 141 CZ ARG A 19 36, .530 32, .120 43 .307 1, .00 57, .69 6

ATOM 142 NE ARG A 19 36, .207 31, .186 42, .351 1, .00 42, .98 7 ATOM 143 CD ARG A 19 37. .338 31. .011 41, .450 1. .00 46, .84 6

ATOM 144 CG ARG A 19 37, .117 31, .155 39 .995 1, .00 33, .34 6

ATOM 145 CB ARG A 19 35 .800 30, .421 39 .724 1, .00 26 .86 6

ATOM 146 CA ARG A 19 35 .773 30, .449 38 .180 1, .00 24, .16 6

ATOM 147 C ARG A 19 34, .635 29, .545 37, .735 1, .00 32, .80 6 ATOM 148 O ARG A 19 34, .691 28. .447 38, .295 1, .00 38, .37 8

ATOM 149 N GLY A 20 33, .659 29. .890 36, .943 1. .00 26, .10 7

ATOM 150 CA GLY A 20 32, .569 28. .978 36, .587 1. .00 22. .13 6

ATOM 151 C GLY A 20 31, .546 28, .912 37, .702 1, .00 34, .41 6

ATOM 152 O GLY A 20 30, .872 27, .856 37, .735 1. .00 28, .59 8 ATOM 153 N ILE A 21 31, .493 29. .934 38, .591 1. .00 29. .96 7

ATOM '154 CD1 ILE A 21 33, .459 29, .632 41, .814 1, .00 41, ,54 6

ATOM 155 CGI ILE A 21 32, ,100 29. ,052 41, .506 1. .00 25, .19 6

ATOM 156 CB ILE A 21 30, ,975 29. .986 41, .122 1. ,00 26. .29 6

ATOM 157 CG2 ILE A 21 29. .844 29. .735 42, .107 1. ,00 19. ,84 6 ATOM 158 CA ILE A 21 30. .460 29. ,794 39. .684 1. ,00 32. ,15 6

ATOM 159 C ILE A 21 29, .284 30. .745 39, .329 1. .00 27. .88 6

ATOM 160 O ILE A 21 29, .528 31. .975 39. .238 1. ,00 25, .54 8

ATOM 161 N PHE A 22 28. .130 30. ,216 39. .043 1. ,00 22. .71 7

ATOM 162 CD2 PHE A 22 28, .593 30. .211 35, .689 1. .00 27, .44 6 ATOM 163 CE2 PHE A 22 29, .621 30, .567 34, .823 1, .00 24, .36 6

ATOM 164 CZ PHE A 22 29, .741 31. .905 34, .446 1. .00 33, .93 6

ATOM 165 CE1 PHE A 22 28. ,872 32. ,884 34. .911 1. ,00 27. ,82 6

ATOM 166 CD1 PHE A 22 27. .870 32. .510 35, .793 1. ,00 28, .92 6

ATOM 167 CG PHE A 22 27. .724 31. ,192 36. .172 1. ,00 28. .03 6 ATOM 168 CB PHE A 22 26. .658 30. ,789 37. .118 1. ,00 24. .21 6

ATOM 169 CA PHE A 22 26, .950 30. .969 38, .613 1. .00 26, .09 6

ATOM 170 C PHE A 22 25. .683 30. .711 39, .409 1. ,00 25, .39 6

ATOM 171 O PHE A 22 24. .665 31. ,302 38. .981 1. ,00 24. .97 8

ATOM 172 N GLY A 23 25, .607 29. .924 40, .467 1. .00 18, .81 7 ATOM 173 CA GLY A 23 24. .363 29. ,724 41. .148 1. ,00 18. .46 6

ATOM 174 C GLY A 23 23, .503 28. .543 40, .757 1. .00 19. .87 6

ATOM 175 O GLY A 23 22, .414 28. .258 41. .288 1. ,00 21, .97 8

ATOM 176 N ASN A 24 24. .176 27. .813 39. .877 1. .00 24. .80 7

ATOM 177 ND2 ASN A 24 24, .193 25, .603 36, .454 1. ,00 54, .67 7 ATOM 178 OD1 ASN A 24 23, .354 24. .090 38. .041 1. ,00 52. .66 8

ATOM 179 CG ASN A 24 24, .034 25, .056 37, .655 1. .00 54, .67 6 ATOM 180 CB ASN A 24 24.770 26.009 38.589 1.00 32..23 6

ATOM 181 CA ASN A 24 23 .593 26 .534 39 .395 1, ,00 25, .92 6

ATOM 182 C ASN A 24 23 .179 25 .638 40 .552 1, .00 25, .32 6

ATOM 183 O ASN A 24 23, .976 25 .322 41 .465 1, .00 30, ,34 8 ATOM 184 N GLY A 25 21. .885 25, .306 40 .580 1, .00 24. .65 7

ATOM 185 CA GLY A 25 21, .465 24 .504 41 .725 1, .00 28, ,29 6

ATOM 186 C GLY A 25 20 .845 25 .160 42 .938 1, .00 26, ,14 6

ATOM 187 O GLY A 25 20 .160 24 .516 43 .717 1 .00 27 .35 8

ATOM 188 N ALA A 26 21, .025 26, .469 43. .065 1. .00 33. .36 7 ATOM 189 CB ALA A 26 21. .389 28, .357 44 .440 1, .00 22, .66 6

ATOM 190 CA ALA A 26 20, .451 27, .216 44, .226 1. .00 21. .52 6

ATOM 191 C ALA A 26 19, .024 27, .532 43 .905 1. .00 18, .32 6

ATOM 192 O ALA A 26 18 .702 27 .928 42 .773 1, .00 24, .15 8

ATOM 193 N ARG A 27 18 .210 27 .375 44 .899 1 .00 19 .06 7 ATOM 194 NH2 ARG A 27 15 .995 22 .073 47 .281 1, .00 46, .56 7

ATOM 195 NH1 ARG A 27 16, .803 22, .004 45, .047 1, .00 39, ,77 7

ATOM 196 CZ ARG A 27 16, .017 22, .485 46, .012 1. .00 48. .33 6

ATOM 197 NE ARG A 27 15, .098 23, .456 45, .820 1. .00 41, .99 7

ATOM 198 CD ARG A 27 15 .075 24 .160 44 .559 1, .00 36, .91 6 ATOM 199 CG ARG A 27 16 .301 25 .064 44. .358 1. .00 29. .21 6

ATOM 200 CB ARG A 27 15 .999 26. .369 45 .132 1, .00 26, ,05 6

ATOM 201 CA ARG A 27 16 .785 27 .590 44 .764 1, .00 19, .90 6

ATOM 202 C ARG A 27 16. ,462 28. ,820 45. .623 1. ,00 24. ,82 6

ATOM 203 O ARG A 27 16, .484 28. .798 46, .855 1. .00 23. .36 8 ATOM 204 N VAL A 28 16 .090 29, .902 44 .963 1. .00 21. .58 7

ATOM 205 CG2 VAL A 28 18, .212 31. .847 44, .971 1. .00 20, .76 6

ATOM 206 CGI VAL A 28 16, .584 33 .595 45 .659 1, .00 24, .41 6

ATOM 207 CB VAL A 28 16 .756 32 .246 44 .948 1, .00 18, .33 6

ATOM 208 CA VAL A 28 15, .821 31, .208 45 .600 1, ,00 20, .58 6 ATOM 209 C VAL A 28 14, .369 31, .568 45, .504 1, .00 16, .41 6

ATOM 210 O VAL A 28 13, .904 31, .628 44. .344 1, .00 22, .07 8

ATOM 211 N ALA A 29 13, .724 31. .792 46, .617 1. .00 15, .89 7

ATOM 212 CB ALA A 29 11, .536 31, .675 47, .718 1, .00 16, .94 6

ATOM 213 CA ALA A 29 12 .322 32 .248 46 .580 1, .00 21 .50 6 ATOM 214 C ALA A 29 12, .353 33, .820 46, .734 1. .00 19, ,32 6

ATOM 215 O ALA A 29 13 .042 34, .312 47, .649 1, .00 19, .70 8

ATOM 216 N VAL A 30 11, .770 34, .530 45, .806 1. .00 18, .83 7

ATOM 217 CG2 VAL A 30 13. .356 36. .406 44. .142 1. ,00 17. .28 6

ATOM 218 CGI VAL A 30 11, .680 38, .150 44, .538 1. ,00 19, ,61 6 ATOM 219 CB VAL A 30 11, .885 36, .649 44, .450 1. .00 19, ,02 6

ATOM 220 CA VAL A 30 11, .590 35. .993 45, .824 1. .00 21. .94 6

ATOM 221 C VAL A 30 10, .211 36. .329 46, .406 1. .00 17. .79 6

ATOM 222 O VAL A 30 9, .239 36. .104 45. .639 1. ,00 16. .80 8

ATOM 223 N LEU A 31 10, .136 36, .740 47, .677 1. ,00 16, .21 7 ATOM 224 CD2 LEU A 31 8, .443 35. .115 51. .734 1. ,00 18. .64 6

ATOM 225 CD1 LEU A 31 9, .392 34, .230 49, .510 1. ,00 18. .41 6

ATOM 226 CG LEU A 31 8. .513 35. .233 50. .228 1. ,00 27. ,95 6

ATOM 227 CB LEU A 31 8, .841 36. .689 49. .787 1. ,00 17. ,41 6

ATOM 228 CA LEU A 31 8, .837 37. .091 48. .332 1. ,00 17. .17 6 ATOM 229 C LEU A 31 8, .609 38, .573 48. .053 1. .00 23. .39 6

ATOM 230 O LEU A 31 9, .245 39, .436 48, .649 1. .00 19, .56 8

ATOM 231 N ASP A 32 7, .756 38, .918 47, .142 1. .00 20, .33 7

ATOM 232 OD2 ASP A 32 8, .509 42. .872 45. .463 1. .00 17, .46 8

ATOM 233 OD1 ASP A 32 10. .355 42. .272 46. .272 1. ,00 18. .58 8 ATOM 234 CG ASP A 32 9, .249 41. .959 45. .903 1. ,00 17. .91 6

ATOM 235 CB ASP A 32 8, .780 40. .509 45. .770 1. .00 17, .55 6

ATOM 236 CA ASP A 32 7. .544 40 .265 46. .640 1. .00 18 .05 6

ATOM 237 C ASP A 32 6 .259 40 .407 45 .874 1. .00 16 .34 6

ATOM 238 O ASP A 32 5, .265 39, .719 46, .233 1. ,00 18, .95 8 ATOM 239 N THR A 33 6, .345 41. .337 44. .922 1. ,00 18. .08 7

ATOM 240 CG2 THR A 33 5, .111 44. .100 44. .539 1. ,00 15, .20 6 ATOM 241 OG1 THR A 33 6,.078 43,.108 42,.626 1..00 15,.34 8

ATOM 242 CB THR A 33 5, .050 42, .995 43, .536 1. .00 17, .62 6

ATOM 243 CA THR A 33 5. .068 41, .559 44 .165 1. .00 19, .10 6

ATOM 244 C THR A 33 4 .876 40, .503 43, .046 1. .00 21, .43 6 ATOM 245 O THR A 33 3 .956 40 .703 42 .210 1. .00 19, .77 8

ATOM 246 N GLY A 34 5 .747 39 .519 42 .979 1. .00 19, .23 7

ATOM 247 CA GLY A 34 5 .694 38 .503 41 .928 1. .00 18, .38 6

ATOM 248 C GLY A 34 6, .872 38, .646 41, .034 1. ,00 17, .22 6

ATOM 249 O GLY A 34 7. .711 39 .459 41 .383 1, .00 18 .99 8 ATOM 250 N ILE A 35 6, .974 37, .882 39, .956 1. .00 17, .46 7

ATOM 251 CD1 ILE A 35 10 .899 35 .757 40 .596 1, .00 15 .13 6

ATOM 252 CGI ILE A 35 9 .791 36 .828 40 .462 1 .00 14 .72 6

ATOM 253 CB ILE A 35 9, .166 36. .970 39, .068 1, ,00 15, .03 6

ATOM 254 CG2 ILE A 35 10 .243 37, .326 38 .068 1, .00 15 .97 6 ATOM 255 CA ILE A 35 8, .048 37. .960 38, .978 1. .00 14, .81 6

ATOM 256 C ILE A 35 7 .360 37, .965 37 .617 1, .00 17 .66 6

ATOM 257 O ILE A 35 6, .554 37, .071 37, .431 1. .00 21, .48 8

ATOM 258 N ALA A 37 7, .565 38, .985 36, .818 1. .00 17, .09 7

ATOM 259 CB ALA A 37 6, .974 40, .415 34. .895 1. .00 19. .79 6 ATOM 260 CA ALA A 37 6, .929 39, .026 35, .522 1. .00 19 .65 6

ATOM 261 C ALA A 37 7. .799 38, ,217 34, .551 1. .00 17 .88 6

ATOM 262 O ALA A 37 9, .037 38, .066 34, .604 1, .00 21 .23 8

ATOM 263 N SER A 38 7 .062 37, .689 33 .589 1, .00 16 .80 7

ATOM 264 OG SER A 38 7, .219 35, .805 30, .632 1, ,00 30. . 69 8 ATOM 265 CB SER A 38 6 .656 36 .129 31 .852 1, .00 24 .32 6

ATOM 266 CA SER A 38 7, .794 36, .946 32, .527 1. .00 20, .02 6

ATOM 267 C SER A 38 8, .554 38, .064 31, .824 1. .00 20, .83 6

ATOM 268 O SER A 38 8, .026 39, .138 31. .556 1. ,00 21. .16 8

ATOM 269 N HIS A 39 9, .788 37. .876 31. .449 1. ,00 16. ,67 7 ATOM 270 CD2 HIS A 39 11, .839 42. .154 31. .855 1. ,00 18. .50 6

ATOM 271 NE2 HIS A 39 12, .849 42. .828 31. .229 1. ,00 17. .78 7

ATOM 272 CE1 HIS A 39 13 .757 41, .990 30, .654 1. ,00 19, .11 6

ATOM 273 ND1 HIS A 39 13, .250 40, .817 30. .899 1. ,00 18, .95 7

ATOM 274 CG HIS A 39 12. .108 40, .809 31, .630 1. ,00 18. .98 6 ATOM 275 CB HIS A 39 11, .359 39, .557 32, .049 1. ,00 18, .97 6

ATOM 276 CA HIS A 39 10, .744 38, .721 30, .858 1. ,00 19, .12 6

ATOM 277 C HIS A 39 11, .775 37. .948 30, .062 1. ,00 17. .80 6

ATOM 278 O HIS A 39 12, .355 37, .014 30, .570 1. .00 20. .73 8

ATOM 279 N PRO A 40 12. .200 38. .418 28, .889 1. ,00 21, .00 7 ATOM 280 CG PRO A 40 12 .293 39, .449 26, .786 1, ,00 21 .21 6

ATOM 281 CD PRO A 40 11, .597 39, .542 28, .113 1. ,00 18, .96 6

ATOM 282 CB PRO A 40 13. .560 38. ,729 26, .913 1. ,00 19, .67 6

ATOM 283 CA PRO A 40 13, .254 37, ,823 28, .100 1, .00 22, .54 6

ATOM 284 C PRO A 40 14. .534 37, .614 28. .909 1. .00 24, .98 6 ATOM 285 O PRO A 40 15 .326 36, .689 28, .538 1. ,00 23, .15 8

ATOM 286 N ASP A 41 14, .864 38. .402 29. .921 1. ,00 21, .23 7

ATOM 287 OD2 ASP A 41 19, .022 40. .411 31, .203 1. ,00 23, .14 8

ATOM 288 OD1 ASP A 41 18, .902 38, .575 30. .179 1. ,00 20. ,45 8

ATOM 289 CG ASP A 41 18, .278 39, .474 30, .706 1. .00 21. .49 6 ATOM 290 CB ASP A 41 16, .801 39, .675 30, .849 1. .00 17, .52 6

ATOM 291 CA ASP A 41 16, .149 38, .300 30, .623 1. .00 18, .20 6

ATOM 292 C ASP A 41 16 .007 37, .531 31, .930 1. .00 16, .57 6

ATOM 293 O ASP A 41 16, .990 37, .609 32. .687 1. ,00 21, .11 8

ATOM 294 N LEU A 42 14 .877 36 .908 32, .100 1. ,00 16 .23 7 ATOM 295 CD2 LEU A 42 15. .154 37. .970 35. .800 1. .00 20. .71 6

ATOM 296 CD1 LEU A 42 12 .728 38, .634 35, .680 1. ,00 18, .04 6

ATOM 297 CG LEU A 42 13, .906 38. .079 34. .940 1. ,00 22, .07 6

ATOM 298 CB LEU A 42 13, .573 36, .743 34. .250 1. ,00 19, .04 6

ATOM 299 CA LEU A 42 14, .688 36, .119 33, .316 1. ,00 18, .11 6 ATOM 300 C LEU A 42 14, .147 34, .706 33, .035 1. ,00 22, .16 6

ATOM 301 O LEU A 42 13 .321 34 .478 32, .117 1. .00 24. .54 8 ATOM 302 N ARG A 43 14,.426 33..731 33,.856 1..00 20.59 7

ATOM 303 NH2 ARG A 43 16 .861 27 .990 36 .107 1, .00 53 .82 7

ATOM 304 NH1 ARG A 43 14 .504 27 .483 36 .114 1, .00 58 .81 7

ATOM 305 CZ ARG A 43 15 .623 27, .968 35 .534 1, .00 59 .96 6 ATOM 306 NE ARG A 43 15 .539 28 .580 34 .285 1, .00 59 .26 7

ATOM 307 CD ARG A 43 14 .187 29 .098 33 .890 1, .00 53 .79 6

ATOM 308 CG ARG A 43 14 .538 30, .144 32 .891 1, .00 38 .80 6

ATOM 309 CB ARG A 43 14, .893 31, .393 33, .636 1. .00 20 .63 6

ATOM 310 CA ARG A 43 13 .780 32 .413 33 .764 1, .00 21 .97 6 ATOM 311 C ARG A 43 13 .120 32 .158 35 .092 1, .00 20 .02 6

ATOM 312 O ARG A 43 13 .858 32, .194 36 .102 1, .00 24 .03 8

ATOM 313 N ILE A 44 11 .867 31 .959 35 .226 1 .00 20 .63 7

ATOM 314 CD1 ILE A 44 8, .902 34, .679 35, .796 1. .00 25 .57 6

ATOM 315 CGI ILE A 44 10, .068 33, .881 36, .368 1. .00 29 .55 6 ATOM 316 CB ILE A 44 9, .746 32, .360 36, .490 1. ,00 24, .21 6

ATOM 317 CG2 ILE A 44 8 .902 31, .922 37 .662 1. .00 21 .80 6

ATOM 318 CA ILE A 44 11 .103 31, .670 36, .445 1. .00 20 .36 6

ATOM 319 C ILE A 44 10, .838 30, .166 36, .550 1. .00 28 .98 6

ATOM 320 O ILE A 44 10, .177 29, .571 35, .695 1. ,00 23 .55 8 ATOM 321 N ALA A 45 11 .322 29, .549 37 .602 1, ,00 27 .19 7

ATOM 322 CB ALA A 45 12, .254 27, .427 38, .711 1. .00 18 .19 6

ATOM 323 CA ALA A 45 11, .176 28, .111 37, .907 1. .00 25 .70 6

ATOM 324 C ALA A 45 9 .799 27, .798 38 .418 1, .00 25 .04 6

ATOM 325 O ALA A 45 9 .394 26, .706 38, .033 1, .00 28 .94 8 ATOM 326 N GLY A 46 9. .044 28, .597 39, .089 1. .00 20 .03 7

ATOM 327 CA GLY A 46 7, .719 28, .282 39, .555 1. .00 16 .95 6

ATOM 328 C GLY A 46 7. .400 29. .295 40. .624 1. ,00 22, .67 6

ATOM 329 O GLY A 46 8, .103 30. .327 40, .564 1. ,00 21 .98 8

ATOM 330 N GLY A 47 6, .408 29. .068 41. .382 1. ,00 22, .31 7 ATOM 331 CA GLY A 47 6. .038 30. ,017 42. .427 1. ,00 21, .33 6

ATOM 332 C GLY A 47 4, .601 29. ,839 42, .841 1. ,00 25, .87 6

ATOM 333 O GLY A 47 3. .918 28. .882 42. .428 1. ,00 25, .43 8

ATOM 334 N ALA A 48 4. .055 30. ,737 43. .620 1. ,00 20, .53 7

ATOM 335 CB ALA A 48 2, .815 29. .944 45, .442 1. ,00 20, .90 6 ATOM 336 CA ALA A 48 2, .713 30. .745 44, .144 1. ,00 20, .50 6

ATOM 337 C ALA A 48 2, .326 32. .203 44. .460 1. ,00 29, .20 6

ATOM 338 O ALA A 48 3, .178 33. .083 44. .532 1. ,00 25, .97 8

ATOM 339 N SER A 49 1, .068 32, .454 44, .688 1. ,00 2 .19 7

ATOM 340 OG SER A 49 -0, .986 35. .495 44, .409 1. ,00 27, .17 8 ATOM 341 CB SER A 49 -0, .441 34. .225 43, .938 1. ,00 26, .70 6

ATOM 342 CA SER A 49 0, .478 33. .712 45, .013 1. ,00 22, .03 6

ATOM 343 C SER A 49 -0, .307 33. .577 46, .315 1. ,00 31, .92 6

ATOM 344 O SER A 49 -1. .067 32. .591 46. .360 1. ,00 26, .97 8

ATOM 345 N PHE A 50 -0. .097 34. .588 47. .147 1. ,00 22, .91 7 ATOM 346 CD2 PHE A 50 -0. .049 32. ,109 50. .111 1. 00 31, .06 6

ATOM 347 CE2 PHE A 50 0. .409 30. .786 49. .993 1. ,00 23, .47 6

ATOM 348 CZ PHE A 50 1. .692 30. .585 49. .509 1. ,00 26, .37 6

ATOM 349 CE1 PHE A 50 2. .459 31. .650 49. .044 1. ,00 27, .36 6

ATOM 350 CD1 PHE A 50 1. .909 32. .920 49, .123 1. ,00 25 .18 6 ATOM 351 CG PHE A 50 0, .659 33. .206 49, .640 1. ,00 27 .18 6

ATOM 352 CB PHE A 50 0, .068 34. .581 49. .654 1. ,00 20, .39 6

ATOM 353 CA PHE A 50 -0 .814 34, .627 48, .416 1. .00 20 .79 6

ATOM 354 C PHE A 50 -1, .699 35, .845 48, .217 1. .00 26 .50 6

ATOM 355 O PHE A 50 -2, .095 36, .380 49, .255 1. .00 33 .21 8 ATOM 356 N ILE A 51 -2. .067 36. ,337 47. .068 1. ,00 25, .81 7

ATOM 357 CD1 ILE A 51 -0. .964 39. ,394 48. .263 1. ,00 25. .15 6

ATOM 358 CGI ILE A 51 -0, .838 39. .160 46, .744 1. ,00 26, .10 6

ATOM 359 CB ILE A 51 -2, .155 38. .659 46, .174 1. ,00 28, .46 6

ATOM 360 CG2 ILE A 51 -2, .994 39. .906 45. .884 1. ,00 26, .35 6 ATOM 361 CA ILE A 51 -2 .870 37. .563 46, .980 1. .00 25 .17 6

ATOM 362 C ILE A 51 -4 .111 37. .059 46, .276 1. .00 22 .13 6 ATOM 363 O ILE A 51 -4.019 36.809 45.075 1.00 26.47 8

ATOM 364 N SER A 52 -5 .211 36 .990 46 .985 1 .00 31 .96 7

ATOM 365 OG SER A 52 -7 .326 37 .187 48 .213 1 .00 55 .96 8

ATOM 366 CB SER A 52 -7 .637 36 .283 47 .168 1 .00 40 .98 6 ATOM 367 CA SER A 52 -6 .416 36 .494 46 .288 1 .00 36 .15 6

ATOM 368 C SER A 52 -6 .840 37 .320 45 .088 1 .00 41 .46 6

ATOM 369 O SER A 52 -7 .334 36 .557 44 .131 1 .00 42 .48 8

ATOM 370 N SER A 53 -6 .711 38 .640 45 .097 1 .00 34 .99 7

ATOM 371 OG SER A 53 -6 .064 41 .220 44 .420 1 .00 45 .24 8 ATOM 372 CB SER A 53 -7 .345 40 .753 44 .027 1 .00 36 .41 6

ATOM 373 CA SER A 53 -7 .166 39 .272 43 .832 1 .00 32 .42 6

ATOM 374 C SER A 53 -6 .198 39 .008 42 .704 1 .00 28 .79 6

ATOM 375 O SER A 53 -6. .518 39, .427 41, .610 1, .00 30 .59 8

ATOM 376 N GLU A 54 -5 .089 38 .335 42 .931 1 .00 26 .60 7 ATOM 377 OE2 GLU A 54 -2. .266 42, .297 42 .536 1, .00 28 .17 8

ATOM 378 OE1 GLU A 54 -0, .866 41, .124 41, .290 1, .00 25 .34 8

ATOM 379 CD GLU A 54 -1 .988 41 .335 41 .716 1, .00 26 .67 6

ATOM 380 CG GLU A 54 -3 .245 40 .511 41 .554 1, .00 33 .12 6

ATOM 381 CB GLU A 54 -2 .993 39 .046 41 .906 1 .00 30 .53 6 ATOM 382 CA GLU A 54 -4 .147 38 .053 41 .836 1 .00 27 .17 6

ATOM 383 C GLU A 54 -3, .550 36, .669 41, .985 1. .00 29, .10 6

ATOM 384 O GLU A 54 -2 .499 36, .360 42, .543 1. .00 31 .16 8

ATOM 385 N PRO A 55 -4, .303 35, .698 41, .531 1, .00 28 .22 7

ATOM 386 CG PRO A 55 -6, .256 34, .510 40. .919 1. .00 32, .87 6 ATOM 387 CD PRO A 55 -5 .638 35 .901 40, .877 1, .00 27 .93 6

ATOM 388 CB PRO A 55 -5 .108 33, .565 40, .980 1. .00 25 .50 6

ATOM 389 CA PRO A 55 -3 .921 34 .295 41 .596 1 .00 27 .69 6

ATOM 390 C PRO A 55 -2 .652 33 .893 40 .869 1, .00 26 .18 6

ATOM 391 O PRO A 55 -2 .111 32, .861 41, .284 1, .00 29 .26 8 ATOM 392 N SER A 57 -2, .177 34, .589 39, .865 1, .00 23 .03 7

ATOM 393 OG SER A 57 0, .204 34, .676 37, ,165 1. .00 24, .28 8

ATOM 394 CB SER A 57 -1, .012 34. .882 37. .811 1. .00 17, .78 6

ATOM 395 CA SER A 57 -0. .933 34. .228 39. .178 1. ,00 17, .61 6

ATOM 396 C SER A 57 0, .231 34, .769 40. .022 1. ,00 23, .28 6 ATOM 397 O SER A 57 0, .077 35. .788 40. .730 1. .00 23, .01 8

ATOM 398 N TYR A 58 1. .401 34, .208 39. .978 1. ,00 21 .42 7

ATOM 399 OH TYR A 58 5, .286 30, .151 36. .865 1. ,00 33 .08 8

ATOM 400 CD2 TYR A 58 4, ,751 33. ,134 38. .858 1. ,00 20, .82 6

ATOM 401 CE2 TYR A 58 5. .242 32. .389 37. .792 1. ,00 27, .67 6 ATOM 402 CZ TYR A 58 4. .847 31. .036 37. .806 1. ,00 30, .71 6

ATOM 403 CE1 TYR A 58 4. .098 30. ,504 38. ,847 1. ,00 24. .64 6

ATOM 404 CD1 TYR A 58 3. ,650 31. .337 39. ,884 1. ,00 30, .01 6

ATOM 405 CG TYR A 58 3, .956 32. .697 39. .898 1. ,00 24, ,45 6

ATOM 406 CB TYR A 58 3, .496 33, .547 41. ,049 1. .00 19, .56 6 ATOM 407 CA TYR A 58 2, .579 34. .707 40. .656 1. .00 22, .41 6

ATOM 408 C TYR A 58 3, ,245 35. .769 39. .795 1. ,00 18, .11 6

ATOM 409 O TYR A 58 4. .272 36. .323 40. ,134 1. ,00 19, .48 8

ATOM 410 N HIS A 59 2. .819 36. ,120 38. ,608 1. ,00 19. .19 7

ATOM 411 CD2 HIS A 59 2, .574 34. .690 35. .084 1. ,00 24. .45 6 ATOM 412 NE2 HIS A 59 3. .570 33. .918 34. .542 1. ,00 23, .56 7

ATOM 413 CE1 HIS A 59 4. .820 34. .391 34. .635 1. ,00 23, .74 6

ATOM 414 ND1 HIS A 59 4. .689 35. .505 35. .318 1. ,00 27, .94 7

ATOM 415 CG HIS A 59 3, .333 35. .753 35. .529 1. .00 23, .77 6

ATOM 416 CB HIS A 59 2, .907 37. .006 36. .276 1. .00 23, .35 6 ATOM 417 CA HIS A 59 3. ,464 37. .096 37. ,717 1. 00 23. .68 6

ATOM 418 C HIS A 59 3. .223 38. ,478 38. .330 1. ,00 16, .77 6

ATOM 419 O HIS A 59 2. .112 38. ,802 38. ,813 1. ,00 20. .69 8

ATOM 420 N ASP A 60 4, .262 39. .225 38. .217 1. .00 17. .78 7

ATOM 421 OD2 ASP A 60 7, .207 42. .684 39. .352 1. ,00 16, .87 8 ATOM 422 OD1 ASP A 60 5, .224 42. .870 40. .299 1. ,00 17, .98 8

ATOM 423 CG ASP A 60 6 .005 42, .319 39. .583 1. .00 15 .82 6 ATOM 424 CB ASP A 60 5.713 41.108 38.718 .00 20.17 6

ATOM 425 CA ASP A 60 4.257 40.615 38.746 .00 19.60 6

ATOM 426 C ASP A 60 3.449 41.628 37.887 ,00 16.78 6

ATOM 427 O ASP A 60 3.755 41.641 36.688 .00 17.17 8

ATOM 428 N ASN A 61 2.553 42.321 38.565 .00 16.17 7

ATOM 429 ND2 ASN A 61 -0.712 41.216 38.409 .00 21.25 7

ATOM 430 OD1 ASN A 61 0.074 41.753 36.354 .00 22.89 8

ATOM 431 CG ASN A 61 -0.126 42.022 37.543 .00 19.95 6

ATOM 432 CB ASN A 61 0.343 43.358 38.057 ,00 18.61 6

ATOM 433 CA ASN A 61 1.837 43.400 37.853 ,00 18.92 6

ATOM 434 C ASN A 61 .346 44.793 38.274 ,00 22.66 6

ATOM 435 O ASN A 61 .893 45.845 37.801 ,00 23.21 8

ATOM 436 N ASN A 62 .297 44.887 39.186 ,00 19.85 7

ATOM 437 ND2 ASN A 62 .761 48.155 42.016 .00 22.91 7

ATOM 438 OD1 ASN A 62 .928 47.387 41.972 .00 21.51 8

ATOM 439 CG ASN A 62 4.708 47.221 41.809 00 24.07 6

ATOM 440 CB ASN A 62 4.074 45.934 41.266 ,00 15.90 6

ATOM 441 CA ASN A 62 .942 46.038 39.781 ,00 17.18 6

ATOM 442 C ASN A 62 .262 46.370 39.149 ,00 21.56 6

ATOM 443 O ASN A 62 .450 47.489 38.652 ,00 23.34 8

ATOM 444 N GLY A 63 .219 45.499 39.274 ,00 16.07 7

ATOM 445 CA GLY A 63 7.560 45.696 38.775 ,00 15.56 6

ATOM 446 C GLY A 63 8.566 45.526 39.928 00 13.16 6

ATOM 447 O GLY A 63 9.705 45.220 39.576 ,00 14.42 8

ATOM 448 N HIS A 64 8.181 45.732 41.170 00 14.55 7

ATOM 449 CD2 HIS A 64 9.944 47.365 45.114 00 19.41 6

ATOM 450 NE2 HIS A 64 10.615 47.068 46.239 ,00 17.69 7

ATOM 451 CE1 HIS A 64 10.371 45.792 46.555 1.00 17.59 6

ATOM 452 ND1 HIS A 64 9.605 45.312 45.607 ,00 19.22 7

ATOM 453 CG HIS A 64 9.334 46.232 44.659 00 17.77 6

ATOM 454 CB HIS A 64 8.428 45.991 43.484 00 13.22 6

ATOM 455 CA HIS A 64 9.195 45.658 42.241 00 17.90 6

ATOM 456 C HIS A 64 9.902 44.259 42.331 00 17.60 6

ATOM 457 O HIS A 64 11.161 44.161 42.393 00 15.99 8

ATOM 458 N GLY A 65 9.081 43.180 42.309 00 16.44 7

ATOM 459 CA GLY A 65 9.616 41.816 42.380 00 14.82 6

ATOM 460 C GLY A 65 10.479 41.481 41.172 00 14.51 6

ATOM 461 O GLY A 65 11.471 40.769 41.349 00 17.10 8

ATOM 462 N THR A 66 10.099 41.938 39.997 00 14.08 7

ATOM 463 CG2 THR A 66 10.799 41.935 36.263 00 16.28 6

ATOM 464 OG1 THR A 66 8.783 41.636 37.548 00 16.38 8

ATOM 465 CB THR A 66 10.092 42.160 37.567 00 13.88 6

ATOM 466 CA THR A 66 10.851 41.608 38.787 00 11.82 6

ATOM 467 C THR A 66 12.223 42.209 38.848 00 17.20 6

ATOM 468 O THR A 66 13.251 41.729 38.360 00 15.82

ATOM 469 N HIS A 67 12.283 43.430 39.440 00 16.72

ATOM 470 CD2 HIS A 67 14.672 47.526 38.936 00 14.06

ATOM 471 NE2 HIS A 67 15.894 48.068 39.341 00 15.93

ATOM 472 CE1 HIS A 67 16.222 47.455 40.502 00 16.28

ATOM 473 ND1 HIS A 67 15.270 46.657 40.870 00 14.20

ATOM 474 CG HIS A 67 14.288 46.658 39.897 00 13.11

ATOM 475 CB HIS A 67 13.142 45.733 40.058 00 13.83

ATOM 476 CA HIS A 67 13.524 44.275 39.602 00 17.85

ATOM 477 C HIS A 67 14.489 43.467 40.555 00 12.74

ATOM 478 O HIS A 67 15.676 43.217 40.217 00 14.79

ATOM 479 N VAL A 68 13.875 43.184 41.692 1.00 15.52

ATOM 480 CG2 VAL A 68 13.554 43.532 44.544 00 16.01

ATOM 481 CGI VAL A 68 14.397 41.111 44.868 00 15.56

ATOM 482 CB VAL A 68 13.732 42.126 43.930 00 17.25

ATOM 483 CA VAL A 68 14.631 42.373 42.702 00 18.13

ATOM 484 C VAL A 68 15.115 41.029 42.063 00 13.97 ATOM 485 O VAL A 68 16.303 40.718 42.241 1.00 15.56 8

ATOM 486 N ALA A 69 14 .226 40 .381 41 .343 1 .00 16 .97 7

ATOM 487 CB ALA A 69 13 .385 38 .483 40 .044 1 .00 15 .14 6

ATOM 488 CA ALA A 69 14 .625 39 .104 40 .683 1 .00 20 .11 6 ATOM 489 C ALA A 69 15 .800 39 .240 39 .746 1 .00 19 .97 6

ATOM 490 O ALA A 69 16 .716 38 .370 39 .765 1 .00 18 .07 8

ATOM 491 N GLY A 70 15 .860 40 .297 38 .929 1 .00 16 .08 7

ATOM 492 CA GLY A 70 16, .915 40 .521 37 .962 1. .00 13 .42 6

ATOM 493 C GLY A 70 18. .248 40 .803 38 .624 1 .00 17 .11 6 ATOM 494 O GLY A 70 19 .301 40 .458 38 .069 1 .00 18 .05 8

ATOM 495 N THR A 71 18 .251 41 .364 39 .834 1 .00 16 .82 7

ATOM 496 CG2 THR A 71 20 .803 42 .713 42 .461 1 .00 11 .71 6

ATOM 497 OG1 THR A 71 19, .044 43 .833 41 .152 1, .00 19 .96 8

ATOM 498 CB THR A 71 19 .494 42 .605 41 .692 1 .00 17 .79 6 ATOM 499 CA THR A 71 19 .570 41 .620 40 .463 1 .00 18 .16 6

ATOM 500 C THR A 71 20, .085 40 .254 40 .907 1, .00 16 .28 6

ATOM 501 O THR A 71 21 .302 40 .022 40 .823 1. .00 20 .35 8

ATOM 502 N ILE A 72 19 .224 39 .377 41 .381 1 .00 17 .87 7

ATOM 503 CD1 ILE A 72 16, .919 37, .403 44 .477 1, .00 15 .03 6 ATOM 504 CGI ILE A 72 18, .141 37, .904 43 .767 1. .00 16, .72 6

ATOM 505 CB ILE A 72 18, .628 37. .243 42 .500 1, .00 22 .03 6

ATOM 506 CG2 ILE A 72 19 .096 35 .809 42 .923 1, .00 18 .50 6

ATOM 507 CA ILE A 72 19 .708 38. .025 41 .767 1, .00 18 .21 6

ATOM 508 C ILE A 72 20, .158 37 .194 40 .536 1, .00 18 .25 6 ATOM 509 O ILE A 72 21, .223 36, .584 40 .501 1, .00 17, .34 8

ATOM 510 N ALA A 73 19, .308 37, .143 39 .514 1, .00 18 .67 7

ATOM 511 CB ALA A 73 18. .850 34, .961 38, .811 1. .00 21, .72 6

ATOM 512 CA ALA A 73 19. .600 36, .258 38 .384 1. .00 20 .55 6

ATOM 513 C ALA A 73 19, .220 36, .650 36 .993 1, .00 21 .64 6 ATOM 514 O ALA A 73 18, .847 35 .677 36 .292 1, .00 21 .02 8

ATOM 515 N ALA A 74 19, ,351 37. .891 36, .551 1. ,00 19, .57 7

ATOM 516 CB ALA A 74 19. ,407 39. .748 34, .855 1. .00 16, .43 6

ATOM 517 CA ALA A 74 19. ,129 38. .268 35, .176 1. .00 17, .51 6

ATOM 518 C ALA A 74 20, .182 37, .387 34, .423 1. .00 21 .22 6 ATOM 519 O ALA A 74 21, .379 37. .294 34, .773 1. .00 18, .12 8

ATOM 520 N LEU A 75 19. .625 36, .759 33, .380 1. .00 19, .89 7

ATOM 521 CD2 LEU A 75 18. ,684 33. .287 32. .938 1. ,00 20, .44 6

ATOM 522 CD1 LEU A 75 17. .370 34, .159 30, .853 1. .00 22, .84 6

ATOM 523 CG LEU A 75 18. ,279 34. .390 32. .036 1. ,00 23. .72 6 ATOM 524 CB LEU A 75 19. ,491 35. .129 31, .487 1. ,00 22, .59 6

ATOM 525 CA LEU A 75 20. .421 35, .799 32, .558 1. ,00 21, .45 6

ATOM 526 C LEU A 75 21. .644 36, .353 31, .885 1. .00 22, .38 6

ATOM 527 O LEU A 75 21. ,691 37. ,506 31. ,413 1. 00 21. ,99 8

ATOM 528 N ASN A 76 22. ,678 35. ,519 31. ,836 1. 00 23. .39 7 ATOM 529 ND2 ASN A 76 27. .453 34. ,761 31. .699 1. .00 31. .91 7

ATOM 530 OD1 ASN A 76 26. .466 36, .466 30, .730 1. .00 26, .97 8

ATOM 531 CG ASN A 76 26. ,339 35, .407 31, .355 1. ,00 33. .84 6

ATOM 532 CB ASN A 76 24. ,992 34. .941 31, .890 1. ,00 18. .81 6

ATOM 533 CA ASN A 76 23. ,966 35. ,823 31. .226 1. .00 22. .81 6 ATOM 534 C ASN A 76 23. ,762 35. ,565 29. .728 1. 00 32. ,51 6

ATOM 535 O ASN A 76 23. 757 34. ,402 29. ,350 1. 00 27. ,52 8

ATOM 536 N ASN A 77 23. ,499 36. ,553 28. .890 1. ,00 29. ,68 7

ATOM 537 ND2 ASN A 77 19. .501 36. .639 28. .267 1. ,00 20, ,91 7

ATOM 538 OD1 ASN A 77 21. .260 38. ,058 28. ,176 1. ,00 23. .61 8 ATOM 539 CG ASN A 77 20. 739 36. ,958 28. ,001 1. 00 23. ,21 6

ATOM 540 CB ASN A 77 21. ,698 36. ,006 27. ,290 1. 00 24. ,11 6

ATOM 541 CA ASN A 77 23. ,184 36. .392 27. .455 1. .00 29. .10 6

ATOM 542 C ASN A 77 23. .597 37. .625 26. .699 1. ,00 23. .45 6

ATOM 543 O ASN A 77 24. ,554 38. .269 27. .092 1. ,00 26. .46 8 ATOM 544 N SER A 78 22. .917 37. .914 25. .631 1. ,00 23. .85 7

ATOM 545 OG SER A 78 23. .826 38. .128 22. .933 1. ,00 51. .66 8 ATOM 546 CB SER A 78 22.726 38.836 23.468 1.00 38.99 6

ATOM 547 CA SER A 78 23.343 39.124 24.902 1.00 28.32 6

ATOM 548 C SER A 78 22.590 40.392 25.196 1.00 26.17 6

ATOM 549 O SER A 78 22.848 41.406 24.556 1.00 30.79 8 ATOM 550 N ILE A 79 21.553 40.260 25.994 1.00 26.87 7

ATOM 551 CD1 ILE A 79 17.234 39.484 26.505 1.00 22.48 6

ATOM 552 CGI ILE A 79 18.723 39.666 26.593 1.00 23.59 6

ATOM 553 CB ILE A 79 19.291 40.851 25.835 1.00 29.56 6

ATOM 554 CG2 ILE A 79 19.401 40.371 24.400 1.00 25.83 6 ATOM 555 CA ILE A 79 20.675 41.390 26.218 1.00 22.47 6

ATOM 556 C ILE A 79 20.590 41.758 27.679 1.00 22.23 6

ATOM 557 O ILE A 79 21.096 41.041 28.498 1.00 21.05 8

ATOM 558 N GLY A 80 19.921 42.847 27.901 00 21.78 7

ATOM 559 CA GLY A 80 19.579 43.296 29.237 00 20.74 6 ATOM 560 C GLY A 80 20.731 43.409 30.215 00 22.30 6

ATOM 561 O GLY A 80 21.767 43.988 29.848 00 24.00 8

ATOM 562 N VAL A 81 20.534 42.884 31.415 00 20.72 7

ATOM 563 CG2 VAL A 81 19.687 43.194 34.148 00 16.10 6

ATOM 564 CGI VAL A 81 20.666 45.283 33.070 00 19.66 6 ATOM 565 CB VAL A 81 20.938 43.844 33.561 00 21.57 6

ATOM 566 CA VAL A 81 21.616 43.067 32.414 00 18.79 6

ATOM 567 C VAL A 81 22.121 41.681 32.721 00 24.48 6

ATOM 568 O VAL A 81 21.953 40.670 32.065 00 22.82 8

ATOM 569 N LEU A 82 22.797 41.495 33.827 00 26.20 7 ATOM 570 CD2 LEU A 82 27.235 39.378 34.412 00 20.59 6

ATOM 571 CD1 LEU A 82 25.342 37.924 33.896 00 22.30 6

ATOM 572 CG LEU A 82 25.740 39.235 34.558 00 22.25 6

ATOM 573 CB LEU A 82 24.947 40.464 34.054 1.00 20.75 6

ATOM 574 CA LEU A 82 23.431 40.297 34.339 1.00 21.39 6 ATOM 575 C LEU A 82 23.171 40.165 35.847 1 00 19.49 6

ATOM 576 O LEU A 82 23.528 41.144 36.502 1 00 23.34 8

ATOM 577 N GLY A 83 22.671 39.066 36.348 1 00 20.69 7

ATOM 578 CA GLY A 83 22.457 38.949 37.770 1 00 17.03 6

ATOM 579 C GLY A 83 23.782 38.468 38.350 1 00 17.15 6 ATOM 580 O GLY A 83 24.759 38.085 37.729 1 00 17.70 8

ATOM 581 N VAL A 84 23.723 38.456 39.683 1 00 21.38 7

ATOM 582 CG2 VAL A 84 24.533 39.699 42.307 1 00 17.59 6

ATOM 583 CGI VAL A 84 25.675 37.585 42.933 1 00 18.61 6

ATOM 584 CB VAL A 84 24.568 38.197 42.032 1 00 19.33 6 ATOM 585 CA VAL A 84 24.791 37.919 40.537 1 00 18.94 6

ATOM 586 C VAL A 84 24.883 36.373 40.292 1 00 19.93 6

ATOM 587 O VAL A 84 26.024 35.890 40.194 1 00 18.82 8

ATOM 588 N ALA A 85 23.766 35.668 40.255 1 00 19.98 7

ATOM 589 CB ALA A 85 23.136 33.645 41.452 1 00 16.16 6 ATOM 590 CA ALA A 85 23.717 34.185 40.149 1 00 23.42 6

ATOM 591 C ALA A 85 22.819 33.819 38.945 1 00 15.76 6

ATOM 592 O ALA A 85 21.669 33.420 39.123 1 00 17.91 8

ATOM 593 N PRO A 86 23.320 34.080 37.739 1 00 19.61 7

ATOM 594 CG PRO A 86 24.802 34.328 35.990 1 00 22.42 6 ATOM 595 CD PRO A 86 24.691 34.594 37.481 1.00 17.62 6

ATOM 596 CB PRO A 86 23.412 34.286 35.395 1.00 18.97 6

ATOM 597 CA PRO A 86 22.527 33.884 36.525 1.00 22.90 6

ATOM 598 C PRO A 86 21.982 32.494 36.282 1.00 25.04 6

ATOM 599 O PRO A 86 21.044 32.392 35.510 1.00 25.03 8 ATOM 600 N SER A 87 22.550 31.531 36.954 1, 00 21.61 7

ATOM 601 OG SER A 87 23.828 29.588 35.364 1 00 24.93 8

ATOM 602 CB SER A 87 23.195 29.132 36.539 1 00 21.86 6

ATOM 603 CA SER A 87 22.079 30.144 36.789 1 00 25.33 6

ATOM 604 C SER A 87 21.253 29.730 37.973 1 00 27.17 6 ATOM 605 O SER A 87 20.806 28.602 37.975 1, 00 26.19

ATOM 606 N ALA A 88 20.892 30.516 38.966 1, 00 23.05 ATOM 607 CB ALA A 88 20.108 31.154 41.189 .00 18.32 6

ATOM 608 CA ALA A 88 20.051 30.084 40.053 .00 22.79 6

ATOM 609 C ALA A 88 18.628 29.760 39.608 .00 21.41 6

ATOM 610 O ALA A 88 18.106 30.259 38.608 .00 25.76 ATOM 611 N ASP A 89 17.896 28.967 40.323 .00 19.89

ATOM 612 OD2 ASP A 89 16.801 26.516 38.434 .00 31.22

ATOM 613 OD1 ASP A 89 17.282 25.428 40.116 .00 44.17

ATOM 614 CG ASP A 89 16.662 26.363 39.689 ,00 32.29 6

ATOM 615 CB ASP A 89 16.007 27.380 40.585 ,00 26.99 6 ATOM 616 CA ASP A 89 16.475 28.764 40.089 ,00 22.99 6

ATOM 617 C ASP A 89 15.649 29.788 40.846 .00 26.13 6

ATOM 618 O ASP A 89 15.605 29.765 42.092 .00 23.54 8

ATOM 619 N LEU A ^" 90 14.876 30.620 40.201 .00 23.36 7

ATOM 620 CD2 LEU A 90 14.764 35.038 38.890 .00 23.98 6 ATOM 621 CD1 LEU A 90 15.677 34.244 41.144 .00 23.31 6

ATOM 622 CG LEU A 90 14.540 34.313 40.203 .00 32.59 6

ATOM 623 CB LEU A 90 14.110 32.873 39.878 .00 29.22 6

ATOM 624 CA LEU A 90 14.041 31.659 40.828 ,00 22.24 6

ATOM 625 C LEU A 90 12.643 31.203 41.002 ,00 19.26 6 ATOM 626 O LEU A 90 12.017 30.724 40.038 ,00 20.76

ATOM 627 N TYR A 91 12.125 31.476 42.174 ,00 17.22

ATOM 628 OH TYR A 91 12.321 25.105 41.504 .00 31.21

ATOM 629 CD2 TYR A 91 10.097 27.804 42.484 .00 24.84

ATOM 630 CE2 TYR A 91 10.565 26.613 41.969 ,00 22.93 ATOM 631 CZ TYR A 91 11.917 26.318 42.020 .00 31.94

ATOM 632 CE1 TYR A 91 12.863 27.261 42.476 .00 23.17 6

ATOM 633 CD1 TYR A 91 12.382 28.442 43.022 .00 19.76 6

ATOM 634 CG TYR A 91 11.026 28.729 43.006 .00 22.41 6

ATOM 635 CB TYR A 91 10.551 30.077 43.551 00 22.69 6 ATOM 636 CA TYR A 91 10.755 31.167 42.437 ,00 17.72 6

ATOM 637 C TYR A 91 10.023 32.465 42.832 ,00 21.10 6

ATOM 638 O TYR A 91 10.483 33.128 43.740 1.00 21.02 8

ATOM 639 N ALA A 92 8.955 32.776 42.133 1.00 23.09 7

ATOM 640 CB ALA A 92 7.352 34.205 40.926 1.00 14.26 6 ATOM 641 CA ALA A 92 8.067 33.911 42.258 1.00 21.27 6

ATOM 642 C ALA A 92 7.090 33.619 43.378 .00 19.16 6

ATOM 643 O ALA A 92 6.104 32.928 43.143 .00 21.07 8

ATOM 644 N VAL A 93 7.184 34.197 44.567 .00 19.51 7

ATOM 645 CG2 VAL A 93 7.656 32.310 46.567 .00 21.27 6 ATOM 646 CGI VAL A 93 5 678 33.194 47.960 .00 19.09 6

ATOM 647 CB VAL A 93 6 745 33.478 46.928 .00 18.62 6

ATOM 648 CA VAL A 93 6 141 34.036 45.629 .00 17.35 6

ATOM 649 C VAL A 93 5, 534 35.446 45.836 .00 18.48 6

ATOM 650 O VAL A 93 6.166 36.320 46.491 .00 17.69 8 ATOM 651 N LYS A 94 4.359 35.587 45.326 .00 15.95 7

ATOM 652 NZ LYS A 94 0.341 38.732 40.786 .00 16.98 7

ATOM 653 CE LYS A 94 1.380 38.435 41.794 .00 17.51 6

ATOM 654 CD LYS A 94 0.902 38.548 43.246 .00 18.13 6

ATOM 655 CG LYS A 94 1 .857 38.317 44.368 00 19.09 6 ATOM 656 CB LYS A 94 2, .668 37.038 44.233 00 16.57 6

ATOM 657 CA LYS A 94 3.611 36.817 45.392 00 21.78 6

ATOM 658 C LYS A 94 3.007 36.982 46.792 .00 25.09 6

ATOM 659 O LYS A 94 1.985 36.358 47.139 ,00 21.82 8

ATOM 660 N VAL A 95 3.600 37.907 47.568 ,00 20.23 7 ATOM 661 CG2 VAL A 95 5.283 38.661 50.019 1.00 20.17 6

ATOM 662 CGI VAL A 95 4.,360 36.294 49.917 1.00 25.66 6

ATOM 663 CB VAL A 95 4.009 37.779 49.976 1.00 30.09 6

ATOM 664 CA VAL A 95 3.030 38.216 48.885 1.00 21.11 6

ATOM 665 C VAL A 95 2.623 39.696 48.987 1.00 24.66 6 ATOM 666 O VAL A 95 2.177 40.080 50.064 1.00 23.19 8

ATOM 667 N LEU A 96 2.818 40.511 47.962 1.00 23.27 7 ATOM 668 CD2 LEU A 96 3.997 43.237 50.138 1.00 25.60 6

ATOM 669 CD1 LEU A 96 5 .970 43 .494 48 .659 1 .00 20 .15 6

ATOM 670 CG LEU A 96 4 .751 42 .698 48 .975 1 .00 22 .84 6

ATOM 671 CB LEU A 96 3 .706 42 .779 47 .891 1 .00 20 .75 6 ATOM 672 CA LEU A 96 2 .451 41 .918 47 .920 1 .00 23 .08 6

ATOM 673 C LEU A 96 1, .703 42, .036 46 .589 1, .00 23 .01 6

ATOM 674 O LEU A 96 2 .061 41, .403 45. .579 1, .00 21 .24 8

ATOM 675 N ASP A 97 0, .689 42, .897 46 .551 1 .00 23 .27 7

ATOM 676 OD2 ASP A 97 2, .600 45, .183 46, .914 1, .00 34 .41 8 ATOM 677 OD1 ASP A 97 0 .584 45 .765 46 .103 1 .00 29 .86 8

ATOM 678 CG ASP A 97 1, .488 44, .950 46 .240 1, .00 30 .57 6

ATOM 679 CB ASP A 97 1 .555 43 .475 45 .731 1 .00 26 .33 6

ATOM 680 CA ASP A 97 0. .137 43. .056 45, .358 1, .00 23, .04 6

ATOM 681 C ASP A 97 0, .478 44, .050 44, .362 1, .00 19 .75 6 ATOM 682 O ASP A 97 1 .581 44, .509 4 .552 1 .00 20 .60 8

ATOM 683 N ARG A 98 0, .293 44, .333 43, .361 1, .00 21 .05 7

ATOM 684 NH2 ARG A 98 6 .414 46 .513 41 .337 1 .00 61 .54 7

ATOM 685 NH1 ARG A 98 5 .383 46, .580 39 .258 1, .00 61 .06 7

ATOM 686 CZ ARG A 98 5, .345 46, .297 40, .563 1, .00 59, .40 6 ATOM 687 NE ARG A 98 4 .287 45 .797 41 .191 1, .00 43 .41 7

ATOM 688 CD ARG A 98 3, .085 45, .642 40, .374 1, .00 30 .97 6

ATOM 689 CG ARG A 98 2 .099 45, .874 41, .477 1, .00 23. .76 6

ATOM 690 CB ARG A 98 0, .838 45, .175 41 .048 1, .00 25 .56 6

ATOM 691 CA ARG A 98 0, .109 45, .190 42, .254 1, .00 25, .82 6 ATOM 692 C ARG A 98 0, .420 46. .628 42, .667 1. .00 23, .93 6

ATOM 693 O ARG A 98 1, .088 47. ,281 41, .838 1, .00 23 .91 8

ATOM 694 N ASN A 99 0, .032 46. .924 43, .851 1. .00 23, .90 7

ATOM 695 ND2 ASN A 99 1, .713 49. .748 42, .838 1, .00 28 .85 7

ATOM 696 OD1 ASN A 99 3, .264 48. .712 44, .128 1. .00 39, .99 8 ATOM 697 CG ASN A 99 2, .098 49, .125 43. .955 1, .00 32 .07 6

ATOM 698 CB ASN A 99 1. ,056 48, ,862 45. .047 1. .00 28. .96 6

ATOM 699 CA ASN A 99 0. .209 48. .265 44, .383 1. .00 30, .38 6

ATOM 700 C ASN A 99 1, .392 48, .195 45, .301 1, .00 30 .88 6

ATOM 701 O ASN A 99 1. .809 49. .252 45, .800 1. .00 30, .12 8 ATOM 702 N GLY A 100 1, .910 47. .022 45. .541 1. .00 24, .81 7

ATOM 703 CA GLY A 100 3, .112 46. .938 46. .388 1. .00 21, .34 6

ATOM 704 C GLY A 100 2. ,730 46. ,700 47. ,825 1. ,00 26. ,62 6

ATOM 705 O GLY A 100 3, .572 46, .651 48. .719 1. .00 30. .05 8

ATOM 706 N SER A 101 1. .455 46. .465 47. .998 1. .00 25. ,04 7 ATOM 707 OG SER A 101 1. .086 47. ,063 50. .195 1. ,00 52. .71 8

ATOM 708 CB SER A 101 0. .288 47. .078 49. .079 1. .00 33. .36 6

ATOM 709 CA SER A 101 1. .004 46. .287 49. .369 1. .00 28. .75 6

ATOM 710 C SER A 101 0. .669 44. ,899 49, .843 1. ,00 37. .54 6

ATOM 711 O SER A 101 0, .182 44. .154 49, .006 1. .00 29, .65 8 ATOM 712 N GLY A 102 0. ,852 44, ,455 51. .064 1. ,00 35, .37 7

ATOM 713 CA GLY A 102 0, ,402 43. .090 51, .473 1. .00 42, .38 6

ATOM 714 C GLY A 102 0. .311 43. .081 53. .009 1. .00 41, .95 6

ATOM 715 O GLY A 102 0, .662 44, .081 53, .674 1. .00 51, .09 8

ATOM 716 N SER A 103 0. ,061 42. ,076 53. .725 1. ,00 30. ,23 7 ATOM 717 OG SER A 103 1. .367 40. .088 54. .944 1. ,00 40. ,84 8

ATOM 718 CB SER A 103 1. .220 41. .179 55, .778 1. .00 31. .04 6

ATOM 719 CA SER A 103 0. .076 41. .926 55, .156 1. .00 29. .72 6

ATOM 720 C SER A 103 1. .057 41. ,013 55. .610 1. ,00 31. .65 6

ATOM 721 O SER A 103 1. .642 40. .294 54. .835 1. .00 34, .54 8 ATOM 722 N LEU A 104 1. ,319 41. ,101 56. ,870 1. 00 28. .22 7

ATOM 723 CD2 LEU A 104 4. ,090 42. ,177 60. ,461 1. ,00 51, .24 6

ATOM 724 CD1 LEU A 104 4. .621 42. .281 58. ,095 1. .00 41. .73 6

ATOM 725 CG LEU A 104 4. .001 41. .439 59. .150 1. ,00 39, .07 6

ATOM 726 CB LEU A 104 2. .654 40. .887 58, .817 1. .00 38 .11 6 ATOM 727 CA LEU A 104 2. .397 40. .307 57. .444 1. .00 31, .06 6

ATOM 728 C LEU A 104 1, .894 38. .866 57. .408 1. ,00 35, .45 6 ATOM 729 O LEU A 104 2.809 38.009 57.345 1.00 34.06 8

ATOM 730 N ALA A 105 0 .578 38 .666 57 .355 1 .00 30 .73 7

ATOM 731 CB ALA A 105 -1 .345 37 .170 57 .302 1 .00 28 .85 6

ATOM 732 CA ALA A 105 0 .171 37 .260 57 .244 1 .00 32 .26 6 ATOM 733 C ALA A 105 0 .492 36 .695 55 .838 1 .00 30 .41 6

ATOM 734 O ALA A 105 0 .790 35 .495 55 .764 1 .00 26 .17 8

ATOM 735 N SER A 106 0 .370 37 .484 54 .767 1 .00 26 .36 7

ATOM 736 OG SER A 106 0 .908 38 .945 52 .353 1 .00 47 .12 8

ATOM 737 CB SER A 106 0 .078 37 .776 52 .335 1 .00 28 .12 6 ATOM 738 CA SER A 106 0 .695 36 .929 53 .429 1 .00 27 .72 6

ATOM 739 C SER A 106 2 .174 36 .648 53 .385 1 .00 24 .51 6

ATOM 740 O SER A 106 2 .586 35 .664 52 .760 1. .00 25 .93 8

ATOM 741 N VAL A 107 3 .021 37 .452 54 .025 1 .00 22 .96 7

ATOM 742 CG2 VAL A 107 5 .113 39 .633 53 .921 1 .00 23 .30 6 ATOM 743 CGI VAL A 107 6, .747 37 .936 54 .918 1 .00 22 .54 6

ATOM 744 CB VAL A 107 5, .292 38 .352 54 .742 1 .00 23 .47 6

ATOM 745 CA VAL A 107 4 .467 37 .209 54 .117 1 .00 22 .96 6

ATOM 746 C VAL A 107 4 .792 35 .863 54 .775 1 .00 27 .46 6

ATOM 747 O VAL A 107 5 .638 35 .148 54 .247 1 .00 22 .03 8 ATOM 748 N ALA A 108 4, .152 35 .572 55 .895 1 .00 26 .22 7

ATOM 749 CB ALA A 108 3, .431 34 .340 57 .872 1 .00 22 .56 6

ATOM 750 CA ALA A 108 4, .291 34, .320 56 .623 1, .00 22, .04 6

ATOM 751 C ALA A 108 3, .862 33, .098 55 .769 1, .00 23, .82 6

ATOM 752 O ALA A 108 4 .541 32 .073 55 .760 1 .00 25 .45 8 ATOM 753 N GLN A 109 2 .798 33 .159 55 .019 1 .00 26 .10 7

ATOM 754 NE2 GLN A 109 -1. .990 31, .648 53, .180 1. .00 56, .51 7

ATOM 755 OE1 GLN A 109 -1, .807 33 .819 52 .964 1, .00 52, .89 8

ATOM 756 CD GLN A 109 -1, .363 32, .789 53 .524 1, .00 52, .62 6

ATOM 757 CG GLN A 109 -0, .163 32, .492 54, .418 1, .00 23, .57 6 ATOM 758 CB GLN A 109 1. .020 32, .469 53, .458 1. .00 19, .24 6

ATOM 759 CA GLN A 109 2. .302 32, .153 54, .141 1. .00 21. .53 6

ATOM 760 C GLN A 109 3. .302 31. .924 53, .060 1. .00 23. ,82 6

ATOM 761 O GLN A 109 3. ,633 30. .801 52, .709 1. ,00 24. ,29 8

ATOM 762 N GLY A 110 3, ,955 32, .956 52, .566 1. .00 26. .56 7 ATOM 763 CA GLY A 110 5. .010 32, .793 51, .539 1. .00 21, .54 6

ATOM 764 C GLY A 110 6. ,193 32, .057 52. .065 1. .00 18. .77 6

ATOM 765 O GLY A 110 6. .890 31. .359 51, .328 1. .00 20. .70 8

ATOM 766 N ILE A 111 6. .506 32. .348 53, .333 1. .00 19. .34 7

ATOM 767 GDI ILE A 111 8. .879 34. .550 56. .483 1. .00 19. ,97 6 ATOM 768 CGI ILE A 111 8. .799 33, .646 55, .221 1. .00 25, ,91 6

ATOM 769 CB ILE A 111 8. .041 32. .300 55, .338 1. .00 21. ,06 6

ATOM 770 CG2 ILE A 111 9. ,069 31. ,422 56. .004 1. ,00 19. ,42 6

ATOM 771 CA ILE A 111 7. ,639 31. .695 54, .014 1. .00 20. ,08 6

ATOM 772 C ILE A 111 7. ,287 30. .164 54. .171 1. ,00 28. ,01 6 ATOM 773 O ILE A 111 8. ,174 29. ,356 53. .925 1. ,00 19. ,72 8

ATOM 774 N GLU A 112 6. ,057 29. .853 54. .534 1. ,00 26. ,23 7

ATOM 775 OΞ2 GLU A 112 5. ,242 26. ,589 57. ,599 1. ,00 55. ,41 8

ATOM 776 OΞ1 GLU A 112 5. 307 28. ,380 59. ,130 1. .00 58. .68 8

ATOM 777 CD GLU A 112 5. 032 27. ,876 57. ,981 1. 00 57. 74 6 ATOM 778 CG GLU A 112 4, ,340 28. .653 56. ,863 1. ,00 54. ,59 6

ATOM 779 CB GLU A 112 4. ,264 28. .406 55. ,355 1. ,00 26. ,07 6

ATOM 780 CA GLU A 112 5. 632 28. ,463 54. .721 1. 00 26. 47 6

ATOM 781 C GLU A 112 5. 651 27. 787 53. ,384 1. 00 24. 57 6

ATOM 782 O GLU A 112 6. ,181 26. ,678 53. ,335 1. ,00 27. ,03 8 ATOM 783 N TRP A 113 5. ,345 28. ,415 52. ,295 1. 00 20. 47 7

ATOM 784 CD2 TRP A 113 5. 939 28. ,229 47. ,577 1. 00 23. 15 6

ATOM 785 CE3 TRP A 113 7. .244 28. .726 47. .644 1. ,00 22. ,83 6

ATOM 786 CZ3 TRP A 113 8. 109 28. ,544 46. ,587 1. 00 22. 30 6

ATOM 787 CH2 TRP A 113 7. 680 27. ,910 45. ,424 1. 00 22. 04 6 ATOM 788 CZ2 TRP A 113 6. 378 27. 441 45. ,332 1. 00 20. 63 6

ATOM 789 CΞ2 TRP A 113 5. .543 27. ,598 46. .399 1. ,00 19. ,44 6 ATOM 790 NE1 TRP A 113 .261 27.215 46.619 1.00 22.83 7

ATOM 791 CD1 TRP A 113 .821 27.559 47.869 1.00 19.44 6

ATOM 792 CG TRP A 113 .847 28.192 48.511 1.00 20.85 6

ATOM 793 CB TRP A 113 .744 28.731 49.896 1.00 20.61 6 ATOM 794 CA TRP A 113 .385 27.849 50.973 1.00 18.92 6

ATOM 795 C TRP A 113 .817 27.518 50.681 1.00 22.62 6

ATOM 796 O TRP A 113 .102 26.484 50.055 1.00 23.67 8

ATOM 797 N ALA A 114 .790 28.387 50.988 1.00 23.59 7

ATOM 798 CB ALA A 114 10.199 29.314 50.947 00 21.64 6 ATOM 799 CA ALA A 114 9.208 28.145 50.684 00 20.45 6

ATOM 800 C ALA A 114 9.720 26.925 51.508 00 24.40 6

ATOM 801 O ALA A 114 10.656 26.271 51.084 00 22.37 8

ATOM 802 N ILE A 115 9.263 26.665 52.696 00 21.85 7

ATOM 803 CD1 ILE A 115 8.887 27.137 57.080 00 21.98 6 ATOM 804 CGI ILE A 115 .735 26.862 55.832 00 22.19 6

ATOM 805 CB ILE A 115 .187 25.725 54.945 00 36.67 6

ATOM 806 CG2 ILE A 115 .445 24.332 55.597 00 26.39 6

ATOM 807 CA ILE A 115 .712 25.557 53.533 00 23.50 6

ATOM 808 C ILE A 115 .183 24.244 52.881 00 22.01 6 ATOM 809 O ILE A 115 .979 23.385 52.509 00 23.68 8

ATOM 810 N ASN A 116 .904 24.294 52.591 00 23.13 7

ATOM 811 ND2 ASN A 116 .718 22.906 53.985 00 35.86 7

ATOM 812 OD1 ASN A 116 .028 23.976 53.170 00 43.01 8

ATOM 813 CG ASN A 116 .117 23.420 52.940 00 31.13 6 ATOM 814 CB ASN A 116 .859 23.287 51.643 00 20.42 6

ATOM 815 CA ASN A 116 .327 23.166 51.910 00 19.55 6

ATOM 816 C ASN A 116 .917 22.893 50.561 00 29.30 6

ATOM 817 O ASN A 116 .758 21.709 50.183 00 30.79 8

ATOM 818 N ASN A 117 8.452 23.795 49.801 00 22.00 7 ATOM 819 ND2 ASN A 117 6.020 24.758 48.002 00 20.38 7

ATOM 820 OD1 ASN A 117 6.621 23.594 46.231 00 25.41 8

ATOM 821 CG ASN A 117 6.944 24.266 47.222 00 21.80 6

ATOM 822 CB ASN A 117 8.400 24.593 47.462 00 19.43 6

ATOM 823 CA ASN A 117 8.993 23.648 48.467 00 18.42 6 ATOM 824 C ASN A 117 10.488 23.572 48.529 00 16.67 6

ATOM 825 O ASN A 117 11.080 23.586 47.448 00 23.59 8

ATOM 826 N ASN A 118 10.994 23.449 49.770 00 24.36 7

ATOM 827 ND2 ASN A 118 14.257 20.977 49.784 00 46.79 7

ATOM 828 OD1 ASN A 118 11.956 20.616 50.768 00 42.51 8 ATOM 829 CG ASN A 118 12.926 20.992 50.037 00 53.99 6

ATOM 830 CB ASN A 118 12.676 22.017 48.931 00 40.09 6

ATOM 831 CA ASN A 118 12.463 23.293 49.763 00 25.20 6

ATOM 832 C ASN A 118 13.436 24.264 49.061 00 29.14 6

ATOM 833 O ASN A 118 14.413 23.816 48.416 00 23.06 ATOM 834 N MET A 119 13.069 25.539 49.345 00 24.91 7

ATOM 835 CE MET A 119 11.345 26.688 45.875 00 25.32 6

ATOM 836 SD MET A 119 12.390 28.044 46.482 00 24.14 16

ATOM 837 CG MET A 119 11.874 27.979 48.232 00 19.15 6

ATOM 838 CB MET A 119 13.167 27.925 49.032 1.00 21.49 6 ATOM 839 CA MET A 119 13.931 26.603 48.812 1.00 20.74 6

ATOM 840 C MET A 119 15.198 26.587 49.594 1.00 19.07 6

ATOM 841 O MET A 119 15.184 26.188 50.752 1.00 23.02 8

ATOM 842 N HIS A 120 16.296 27.065 49.124 1.00 18.99 7

ATOM 843 CD2 HIS A 120 18.647 24.610 49.083 00 30.88 6 ATOM 844 NΞ2 HIS A 120 18.706 23.671 48.118 00 24.71 7

ATOM 845 CE1 HIS A 120 18.992 24.314 46.957 00 28.15 6

ATOM 846 ND1 HIS A 120 19.000 25.611 47.103 00 29.83 7

ATOM 847 CG HIS A 120 18.816 25.840 48.415 00 26.54 6

ATOM 848 CB HIS A 120 18.805 27.181 49.043 00 20.56 6 ATOM 849 CA HIS A 120 17.517 27.249 49.902 00 19.50 6

ATOM 850 C HIS A 120 17.618 28.675 50.536 00 24.81 6 ATOM 851 O HIS A 120 18.213 28.839 51.568 1,.00 18,.22 8

ATOM 852 N ILE A 121 17 .096 29, .668 49 .807 1, .00 20, .09 7

ATOM 853 CD1 ILE A 121 20 .650 31 .060 48 .208 1 .00 18 .27 6

ATOM 854 CGI ILE A 121 19 .750 31 .034 49 .431 1, .00 19 .89 6 ATOM 855 CB ILE A 121 18 .384 31 .719 49 .200 1, .00 24 .43 6

ATOM 856 CG2 ILE A 121 18 .411 33 .247 49 .285 1, .00 19 .92 6

ATOM 857 CA ILE A 121 17 .296 31, .108 50 .101 1, .00 27 .30 6

ATOM 858 C ILE A 121 15 .996 31, .862 49 .892 1, .00 18, .34 6

ATOM 859 O ILE A 121 15 .345 31 .498 48 .913 1, .00 21 .09 8 ATOM 860 N ILE A 122 15 .641 32. .603 50 .895 1, .00 16 .71 7

ATOM 861 CD1 ILE A 122 11 .953 31, .536 53 .181 1, .00 22 .89 6

ATOM 862 CGI ILE A 122 12 .837 31, .911 51 .979 1, .00 24, .32 6

ATOM 863 CB ILE A 122 13 .522 33, .267 52 .001 1. .00 22, .36 6

ATOM 864 CG2 ILE A 122 12 .472 34, .387 52 .058 1. .00 22, .28 6 ATOM 865 CA ILE A 122 14 .414 33 .410 50 .792 1, .00 17 .89 6

ATOM 866 C ILE A 122 14 .873 34, .891 50 .714 1, .00 20 .53 6

ATOM 867 O ILE A 122 15 .632 35, .335 51 .596 1, .00 18 .10 8

ATOM 868 N ASN A 123 14 .457 35, .638 49 .735 1. .00 24, .14 7

ATOM 869 ND2 ASN A 123 14 .634 39, .722 47. .933 1. .00 17, .66 7 ATOM 870 OD1 ASN A 123 16, .741 39, .208 47, .968 1. .00 16, .54 8

ATOM 871 CG ASN A 123 15 .601 38 .839 48 .002 1, .00 18 .32 6

ATOM 872 CB ASN A 123 15 .217 37, .352 48 .089 1, .00 17. .61 6

ATOM 873 CA ASN A 123 14 .771 37, .063 49 .516 1, .00 16, .49 6

ATOM 874 C ASN A 123 13 .519 37, .846 49, .924 1. .00 16, .16 6 ATOM 875 O ASN A 123 12 .473 37, .561 49, .364 1. .00 15, .99 8

ATOM 876 N MET A 124 13, .682 38. .631 51, .003 1. ,00 17. .91 7

ATOM 877 CE MET A 124 12 .625 37, .065 55 .122 1. ,00 18, .43 6

ATOM 878 SD MET A 124 10 .961 37, .279 54 .473 1. .00 25, .22 16

ATOM 879 CG MET A 124 11 .393 37, .747 52 .785 1. .00 22, .98 6 ATOM 880 CB MET A 124 12 .092 39, .072 52, .786 1. .00 16, .34 6

ATOM 881 CA MET A 124 12, .517 39, .473 51, .413 1. .00 19, .71 6

ATOM 882 C MET A 124 12, .848 40. .994 51, .279 1. .00 22. .29 6

ATOM 883 O MET A 124 13 .425 41, .612 52. .209 1. .00 17, .93 8

ATOM 884 N SER A 125 12, .669 41, .567 50, .101 1. .00 19, .47 7 ATOM 885 OG SER A 125 14, ,523 42. .940 48, .182 1. .00 18, .33 8

ATOM 886 CB SER A 125 13, .198 43. .275 48, .457 1. ,00 15. .97 6

ATOM 887 CA SER A 125 12, .942 43. .032 49, .909 1. ,00 18. .46 6

ATOM 888 C SER A 125 11, .655 43. ,750 50, .350 1. ,00 20. .28 6

ATOM 889 O SER A 125 10, .902 44. .316 49, .570 1. .00 19, .39 8 ATOM 890 N LEU A 126 11, .297 43. .695 51, .624 1. .00 17, ,62 7

ATOM 891 CD2 LEU A 126 8, .102 40. .862 51, .658 1. .00 24. .63 6

ATOM 892 CD1 LEU A 126 8 .622 41, .714 53, .877 1. .00 23, .93 6

ATOM 893 CG LEU A 126 8. .997 41, ,757 52. .422 1. ,00 25. ,53 6

ATOM 894 CB LEU A 126 8. .916 43. ,187 51. .871 1. ,00 28. ,42 6 ATOM 895 CA LEU A 126 10. .051 44. ,199 52. .184 1. 00 26. ,68 6

ATOM 896 C LEU A 126 10. .270 44. ,487 53, ,671 1. ,00 21. ,12 6

ATOM 897 O LEU A 126 11. .254 44. .020 54. ,240 ^"1. ,00 20. .64 8

ATOM 898 N GLY A 127 9 .505 45. .329 54, .335 1. .00 22, .92 7

ATOM 899 CA GLY A 127 9, .794 45. .637 55. .735 1. 00 23. ,96 6 ATOM 900 C GLY A 127 8. .602 46. ,346 56. ,347 1. 00 29. ,15 6

ATOM 901 O GLY A 127 7. .718 46. ,926 55. ,745 1. 00 30. ,52 8

ATOM 902 N SER A 128 8, .499 46. .244 57. .635 1. ,00 22. .96 7

ATOM 903 OG SER A 128 5, .648 45. .725 59. .563 1. ,00 44. .80 8

ATOM 904 CB SER A 128 6 .579 45. .564 58, .544 1. ,00 31. .06 6 ATOM 905 CA SER A 128 7, .422 46. .809 58. .423 1. ,00 26. .75 6

ATOM 906 C SER A 128 8, .089 47. .306 59. .704 1. ,00 29. .54 6

ATOM 907 O SER A 128 9. .118 46. ,792 60. .156 1. ,00 25. .89 8

ATOM 908 N THR A 129 7, .438 48. .299 60, .299 1. ,00 33. .31 7

ATOM 909 CG2 THR A 129 7, .743 51. .258 60. .493 1. ,00 30. .94 6 ATOM 910 OG1 THR A 129 6 .191 50, .069 61, .840 1. .00 40, .54 8

ATOM 911 CB THR A 129 7, .555 50. .360 61. .680 1. ,00 32. .41 6 ATOM 912 CA THR A 129 8.018 48.915 61.506 1..00 32.74 6

ATOM 913 C THR A 129 7 .714 48 .005 62 .673 1 .00 32 .62 6

ATOM 914 O THR A 129 8, .427 48 .117 63 .667 1, .00 36, .81 8

ATOM 915 N SER A 130 6, .757 47 .138 62 .480 1, .00 30, .40 7 ATOM 916 OG SER A 130 4 .251 46 .613 62 .921 1, .00 60, .10 8

ATOM 917 CB SER A 130 5. .130 46 .585 64 .070 1 .00 57 .43 6

ATOM 918 CA SER A 130 6 .491 46 .151 63 .545 1, .00 33. .34 6

ATOM 919 C SER A 130 6 .372 44 .754 62 .914 1 .00 41 .55 6

ATOM 920 O SER A 130 6 .086 44 .558 61 .706 1. .00 40 .64 8 ATOM 921 N GLY A 131 6 .541 43 .773 63 .782 1, .00 36, .39 7

ATOM 922 CA GLY A 131 6 .503 42 .373 63 .329 1, .00 34, .64 6

ATOM 923 C GLY A 131 5 .234 41 .724 63 .822 1, .00 35 .04 6

ATOM 924 O GLY A 131 4 .273 42 .468 64 .031 1, .00 42 .75 8

ATOM 925 N SER A 132 5 .179 40 .422 63 .893 1 .00 37 .30 7 ATOM 926 OG SER A 132 3 .196 38 .497 62 .627 1 .00 39 .73 8

ATOM 927 CB SER A 132 2 .876 39 .643 63 .376 1 .00 35 .53 6

ATOM 928 CA SER A 132 3 .986 39 .723 64 .382 1, .00 30, .05 6

ATOM 929 C SER A 132 4 .556 38 .374 64 .813 1, .00 31 .11 6

ATOM 930 O SER A 132 5, .572 37 .836 64 .411 1, .00 32 .00 8 ATOM 931 N SER A 133 3 .842 37, .734 65 .695 1. .00 32, . 96 7

ATOM 932 OG SER A 133 2, .307 36. .218 67. .376 1. .00 54. .62 8

ATOM 933 CB SER A 133 3, .700 36. .342 67, .576 1. .00 47, .70 6

ATOM 934 CA SER A 133 4. .331 36. .440 66. .195 1. ,00 35, .90 6

ATOM 935 C SER A 133 4, .149 35. .380 65, .111 1. ,00 39. .43 6 ATOM 936 O SER A 133 4. .847 34. .366 65. .010 1. ,00 33. .00 8

ATOM 937 N THR A 134 3, .180 35. .667 64. .251 1. ,00 37. .16 7

ATOM 938 CG2 THR A 134 1, .470 34. ,464 61. .014 1. ,00 42. .89 6

ATOM 939 OG1 THR A 134 0, .694 35. .406 63, .113 1. ,00 55. .08 8

ATOM 940 CB THR A 134 1, .813 35. .282 62. .246 1. ,00 54. .29 6 ATOM 941 CA THR A 134 2 .940 34. .724 63. .144 1. .00 39, .11 6

ATOM 942 C THR A 134 4. .213 34. ,729 62. ,288 1. 00 34, ,90 6

ATOM 943 O THR A 134 4, .693 33. .638 61. .945 1. .00 31. ,77 8

ATOM 944 N LEU A 135 4, .600 35. .994 62. .058 1. ,00 30. .88 7

ATOM 945 CD2 LEU A 135 7, .189 39. .568 59. .758 1. .00 28. .02 6 ATOM 946 CD1 LEU A 135 7, .086 37, .378 58. .627 1. ,00 30. .72 6

ATOM 947 CG LEU A 135 7, .166 38, .073 59. .953 1. ,00 28, .29 6

ATOM 948 CB LEU A 135 5. .946 37. .672 60. .799 1. ,00 30, .19 6

ATOM 949 CA LEU A 135 5, .796 36. .201 61. .203 1. ,00 29. .37 6

ATOM 950 C LEU A 135 7, .077 35. .635 61. .777 1. ,00 27. .29 6 ATOM 951 O LEU A 135 7, .958 35. .025 61. ,154 1. ,00 28, .89 8

ATOM 952 N GLU A 136 7, .230 35. ,860 63. ,081 1. ,00 28. .97 7

ATOM 953 OE2 GLU A 136 8, .946 36. .631 67. ,630 1. ,00 43, .17 8

ATOM 954 OE1 GLU A 136 11, .229 36. .190 67. ,562 1. ,00 56, .47 8

ATOM 955 CD GLU A 136 10, .073 36. .210 67. .125 1. ,00 51, .60 6 ATOM 956 CG GLU A 136 9. .871 35. ,664 65. 729 1. 00 37. .60 6

ATOM 957 CB GLU A 136 8, .518 35. .957 65. ,118 1. ,00 30, .71 6

ATOM 958 CA GLU A 136 8. .494 35. ,399 63. ,696 1. ,00 25, .88 6

ATOM 959 C GLU A 136 8, .483 33. ,887 63. ,747 1. ,00 25, .04 6

ATOM 960 O GLU A 136 9 .527 33. .244 63. .636 1. .00 29, .33 8 ATOM 961 N LEU A 137 7, .287 33, ,373 64. .028 1. ,00 26, .02 7

ATOM 962 CD2 LEU A 137 4, .017 29. .845 64. .773 1. ,00 54, .85 6

ATOM 963 CD1 LEU A 137 6, .341 29. ,084 65. ,226 1. ,00 54, .59 6

ATOM 964 CG LEU A 137 5. .487 30. ,026 64. ,399 1. 00 53. .18 6

ATOM 965 CB LEU A 137 5, .909 31. .481 64. ,461 1. ,00 42, .31 6 ATOM 966 CA LEU A 137 7, .330 31. .886 64. ,051 1. ,00 29, ,41 6

ATOM 967 C LEU A 137 7 .745 31, .326 62. .696 1. ,00 31, .98 6

ATOM 968 O LEU A 137 8, .491 30, .301 62. .593 1. ,00 32, .69 8

ATOM 969 N ALA A 138 7. .170 31, .984 61. .676 1. .00 27 .96 7

ATOM 970 CB ALA A 138 6, .457 32. .131 59. .296 1. 00 25. .09 6 ATOM 971 CA ALA A 138 7, .450 31. .547 60. .295 1. ,00 24, .67 6

ATOM 972 C ALA A 138 8, .946 31. .641 60. .041 1. ,00 26, .68 6 ATOM 973 O ALA A 138 9.651 30.736 59.482 1.00 24.43 8

ATOM 974 N VAL A 139 9 .509 32 .777 60 .481 1 .00 24 .43 7

ATOM 975 CG2 VAL A 139 10 .805 35 .468 59 .644 1 .00 23 .18 6

ATOM 976 CGI VAL A 139 12 .736 34 .458 60 .955 1 .00 25 .26 6 ATOM 977 CB VAL A 139 11 .240 34 .427 60 .639 1 .00 23 .72 6

ATOM 978 CA VAL A 139 10 .946 32 .963 60 .179 1 .00 24 .64 6

ATOM 979 C VAL A 139 11 .785 31 .875 60 .847 1 .00 22 .27 6

ATOM 980 O VAL A 139 12 .734 31 .316 60 .296 1 .00 24 .72 8

ATOM 981 N ASN A 140 11 .486 31 .593 62 .118 1 .00 27 . 66 7 ATOM 982 ND2 ASN A 140 11 .683 32 .285 66 .008 1 .00 42 .32 7

ATOM 983 OD1 ASN A 140 13 .425 32 .414 64 .611 1 .00 36 .78 8

ATOM 984 CG ASN A 140 12 .388 31 .851 64 .974 1 .00 40 .71 6

ATOM 985 CB ASN A 140 11 .762 30 .648 64 .308 1 .00 38 .24 6

ATOM 986 CA ASN A 140 12 .215 30 .570 62 .870 1. .00 28 .09 6 ATOM 987 C ASN A 140 12 .048 29 .142 62 .314 1 .00 23 .74 6

ATOM 988 O ASN A 140 13 .079 28 .438 62 .234 1 .00 27 .56 8

ATOM 989 N ARG A 141 10 .819 28 .818 61 .934 1 .00 29 .30 7

ATOM 990 NH2 ARG A 141 6 .667 24 .020 60 .976 1 .00 62 .35 7

ATOM 991 NH1 ARG A 141 7 .366 25 .245 59 .341 1, .00 62 .64 7 ATOM 992 CZ ARG A 141 6 .619 25 .314 60. .452 1, .00 61, .47 6

ATOM 993 NE ARG A 141 6 .129 26 .266 61 .285 1, .00 59 .61 7

ATOM 994 CD ARG A 141 6, .849 27 .392 61. .861 1, .00 48, .63 6

ATOM 995 CG ARG A 141 8 .296 26 .951 62, .044 1. .00 33, .19 6

ATOM 996 CB ARG A 141 9 .203 27 .214 60 .872 1, .00 26 .71 6 ATOM 997 CA ARG A 141 10, .629 27, .489 61. .338 1, ,00 24. .61 6

ATOM 998 C ARG A 141 11, .475 27, .428 60, .116 1. .00 28, .36 6

ATOM 999 O ARG A 141 12, .111 26, .409 59, .919 1. ,00 30. .57 8

ATOM 1000 N ALA A 142 11 .510 28 .420 59 .220 1, .00 28 .76 7

ATOM 1001 CB ALA A 142 12 .125 29 .617 57, .121 1, .00 22, .79 6 ATOM 1002 CA ALA A 142 12 .326 28 .336 57 .992 1. .00 22, .45 6

ATOM 1003 C ALA A 142 13, .799 28, .193 58. .312 1. .00 23. .46 6

ATOM 1004 O ALA A 142 14, .580 27, .473 57. .674 1. .00 26. .21 8

ATOM 1005 N ASN A 143 14, .220 28, .995 59, .297 1. .00 27, .87 7

ATOM 1006 ND2 ASN A 143 17. .784 30, .625 61. .839 1. ,00 41. . 96 7 ATOM 1007 OD1 ASN A 143 18, .187 30, .679 59, .745 1. .00 34. .24 8

ATOM 1008 CG ASN A 143 17. .322 30, .588 60. .596 1. ,00 29. .11 6

ATOM 1009 CB ASN A 143 15. .871 30. .329 60. .523 1. ,00 29, ,69 6

ATOM 1010 CA ASN A 143 15. ,635 29. .021 59. ,743 1. 00 30. ,16 6

ATOM 1011 C ASN A 143 15. .953 27. .666 60. .335 1. ,00 30. ,12 6 ATOM 1012 O ASN A 143 17. .010 27. .136 59. .946 1. ,00 31. ,87 8

ATOM 1013 N ASN A 144 15, .008 27. .125 61. .112 1. ,00 29. ,34 7

ATOM 1014 ND2 ASN A 144 15. .977 26. .890 64. .048 1. ,00 45. ,86 7

ATOM 1015 OD1 ASN A 144 13. .874 26. .581 64. ,829 1. 00 57. ,91 8

ATOM 1016 CG ASN A 144 14. ,771 26. ,309 63. ,974 1. 00 57. 11 6 ATOM 1017 CB ASN A 144 14. ,450 25. .359 62. ,806 1. 00 44. ,00 6

ATOM 1018 CA ASN A 144 15. ,299 25. ,781 61. ,618 1. 00 29. ,75 6

ATOM 1019 C ASN A 144 15. .282 24. .762 60. ,497 1. ,00 40. ,41 6

ATOM 1020 O ASN A 144 15. .968 23. .716 60. ,573 1. 00 42. ,44 8

ATOM 1021 N ALA A 145 14. ,528 25. ,050 59. ,457 1. 00 34. .04 7 ATOM 1022 CB ALA A 145 13. ,330 24. ,281 57. 390 1. 00 26. 85 6

ATOM 1023 CA ALA A 145 14. ,483 24. ,121 58. ,327 1. 00 20. ,42 6

ATOM 1024 C ALA A 145 15. ,731 24. .288 57. ,552 1. ,00 23. ,85 6

ATOM 1025 O ALA A 145 15. .664 23. .663 56. ,514 1. 00 30. ,91 8

ATOM 1026 N GLY A 146 16, .740 25. .040 57. .840 1. ,00 26. .51 7 ATOM 1027 CA GLY A 146 17, ,921 25. .100 56. ,958 1. 00 22. ,88 6

ATOM 1028 C GLY A 146 17, ,767 26. .214 55. .904 1. 00 27. ,41 6

ATOM 1029 O GLY A 146 18. .735 26. ,130 55. .122 1. 00 24. ,39 8

ATOM 1030 N ILE A 147 16. .707 27. .049 55. .889 1. ,00 21. ,34 7

ATOM 1031 CD1 ILE A 147 13. .320 27. .096 53. .722 1. 00 23. ,01 6 ATOM 1032 CGI ILE A 147 14, .789 27. .060 54. .041 1. ,00 22. .99 6

ATOM 1033 CB ILE A 147 15, .321 28. ,439 54. 332 1. 00 26. 62 6 ATOM 1034 CG2 ILE A 147 15.232 29.384 53.135 1.00 23.14 6

ATOM 1035 CA ILE A 147 16 .730 28 .111 54 .845 1 .00 26 .00 6

ATOM 1036 C ILE A 147 17 .500 29 .398 55 .235 1 .00 18 .99 6

ATOM 1037 O ILE A 147 17. .385 29 .727 56 .411 1 .00 20, .20 8 ATOM 1038 N LEU A 148 18 .230 30 .007 54 .320 1 .00 20, .50 7

ATOM 1039 CD2 LEU A 148 21, .996 32, .963 53 .094 1, .00 21, .59 6

ATOM 1040 CD1 LEU A 148 21, .187 32 .871 55 .483 1 .00 21, .52 6

ATOM 1041 CG LEU A 148 20, .849 32. .729 54 .004 1, .00 21, .01 6

ATOM 1042 CB LEU A 148 20 .076 31 .416 53 .699 1 .00 21 .28 6 ATOM 1043 CA LEU A 148 18 .874 31 .288 54 .622 1 .00 18 .16 6

ATOM 1044 C LEU A 148 17 .890 32 .403 54 .204 1 .00 21 .69 6

ATOM 1045 O LEU A 148 17 .385 32 .443 53 .053 1 .00 18 .87 8

ATOM 1046 N LEU A 149 17, .504 33, .244 55 .115 1, .00 19, .79 7

ATOM 1047 CD2 LEU A 149 13 .039 33 .698 56 .361 1, .00 21, .21 6 ATOM 1048 CD1 LEU A 149 14, .937 32, .303 57, .044 1, .00 29, .79 6

ATOM 1049 CG LEU A 149 14 .430 33 .273 55 .986 1, .00 23, .63 6

ATOM 1050 CB LEU A 149 15, .412 34, .443 55 .914 1, .00 19, .13 6

ATOM 1051 CA LEU A 149 16, .580 34, .382 54, .989 1, .00 18, ,47 6

ATOM 1052 C LEU A 149 17 .403 35 .669 54 .993 1, ,00 22, .25 6 ATOM 1053 O LEU A 149 18, .294 35, .913 55, .802 1, ,00 19, .26 8

ATOM 1054 N VAL A 150 17, .140 36, .501 53, .974 1. .00 21, .30 7

ATOM 1055 CG2 VAL A 150 19, .747 36. .476 52. .518 1. ,00 19. .59 6

ATOM 1056 CGI VAL A 150 19, .570 38, .785 52. .177 1. ,00 22, .93 6

ATOM 1057 CB VAL A 150 18, .710 37, .578 52, .402 1, .00 20, .01 6 ATOM 1058 CA VAL A 150 17, .846 37, .764 53, .660 1, .00 20, .55 6

ATOM 1059 C VAL A 150 16 .751 38 .844 53, .547 1, .00 18, .11 6

ATOM 1060 O VAL A 150 15, .817 38, .657 52, .756 1, .00 18, .48 8

ATOM 1061 N GLY A 151 16 .896 39 .886 54, .338 1, .00 16, .89 7

ATOM 1062 CA GLY A 151 15, .849 40, .980 54, .289 1, .00 20, .73 6 ATOM 1063 C GLY A 151 16, .402 42. .404 54. .347 1, .00 16. .63 6

ATOM 1064 O GLY A 151 17. .563 42. .678 54. .734 1. .00 16. .14 8

ATOM 1065 N ALA A 152 15, .614 43. .322 53. .807 1. .00 17. .20 7

ATOM 1066 CB ALA A 152 14. .900 45. .297 52. .755 1. ,00 14. .94 6

ATOM 1067 CA ALA A 152 15, .998 44. .737 53. .682 1. .00 14. .71 6 ATOM 1068 C ALA A 152 15. .895 45, .381 55, .071 1, .00 13, .99 6

ATOM 1069 O ALA A 152 14, .892 45, .173 55, .788 1, .00 17, .68 8

ATOM 1070 N ALA A 153 16, .952 46, .133 55, .387 1, .00 16. .31 7

ATOM 1071 CB ALA A 153 18. .293 47. .552 56, .901 1. .00 17. .15 6

ATOM 1072 CA ALA A 153 16. .956 46. .875 56. .681 1. .00 16, .19 6 ATOM 1073 C ALA A 153 15. .860 47. .945 56. .800 1. .00 22. .55 6

ATOM 1074 O ALA A 153 15. ,313 48. .113 57. ,913 1. ,00 22. ,09 8

ATOM 1075 N GLY A 154 15. .484 48. .543 55. .690 1. ,00 16. .09 7

ATOM 1076 CA GLY A 154 14. .427 49. ,555 55. ,683 1. ,00 18. .21 6

ATOM 1077 C GLY A 154 15. .049 50. .809 55. .066 1. .00 14. .46 6 ATOM 1078 O GLY A 154 16. .263 50. ,930 54. ,899 1. .00 16. ,40 8

ATOM 1079 N ASN A 155 14, .113 51. .674 54. ,663 1. ,00 20, .62 7

ATOM 1080 ND2 ASN A 155 13, .511 51. .960 50. ,428 1. ,00 16. .52 7

ATOM 1081 ODl ASN A 155 15. .360 51. ,538 51. ,718 1. ,00 19. ,81 8

ATOM 1082 CG ASN A 155 14, .233 52. .033 51. .537 1. ,00 17. .87 6 ATOM 1083 CB ASN A 155 13. .765 52. .902 52. ,677 1. ,00 18. ,24 6

ATOM 1084 CA ASN A 155 14, .551 52. .936 53. ,989 1. ,00 17. .90 6

ATOM 1085 C ASN A 155 14, .159 54. .123 54. ,891 1. ,00 24. ,83 6

ATOM 1086 O ASN A 155 13, .733 55. .098 54. .292 1. ,00 22. ,47 8

ATOM 1087 N THR A 156 14, .154 53. .978 56. .193 1. ,00 20. .39 7 ATOM 1088 CG2 THR A 156 12. .287 53. ,113 58. ,276 1. ,00 23. ,08 6

ATOM 1089 OG1 THR A 156 14. .307 54. .076 59. ,118 1. .00 23. ,01 8

ATOM 1090 CB THR A 156 13. .124 54. ,367 58. ,402 1. ,00 23. ,69 6

ATOM 1091 CA THR A 156 13. .714 54. .997 57. ,116 1. ,00 24. ,79 6

ATOM 1092 C THR A 156 14. .848 56. .011 57. ,320 1. ,00 29. .93 6 ATOM 1093 O THR A 156 14, .402 57. .042 57. .813 1. ,00 27. . 99 8

ATOM 1094 N GLY A 157 16, .086 55, .856 57. .005 1. .00 20, .16 7 ATOM 1095 CA GLY A 157 17.154 56.785 57.245 1.00 25.10 6

ATOM 1096 C GLY A 157 17 .486 57 .000 58 .723 1 .00 29 .14 6

ATOM 1097 O GLY A 157 18 .377 57 .810 58 .961 1 .00 33 .04 8

ATOM 1098 N ARG A 160 16 .904 56 .334 59 .657 1 .00 25 .62 7 ATOM 1099 NH2 ARG A 160 10 .330 58 .682 62 .645 1 .00 60 .34 7

ATOM 1100 NH1 ARG A 160 12 .170 59 .527 63 .732 1, .00 59 .53 7

ATOM 1101 CZ ARG A 160 11 .711 58 .643 62 .754 1 .00 59 .28 6

ATOM 1102 NE ARG A 160 12 .583 57 .864 61 .970 1, .00 57 .95 7

ATOM 1103 CD ARG A 160 13 .994 58 .266 62 .165 1 .00 51 .11 6 ATOM 1104 CG ARG A 160 15 .060 57 .898 61 .220 1, .00 42 .14 6

ATOM 1105 CB ARG A 160 15 .570 56 .502 61 .634 1 .00 31 .02 6

ATOM 1106 CA ARG A 160 17 .041 56 .392 61 .112 1, .00 28 .11 6

ATOM 1107 C ARG A 160 17 .381 55 .048 61 .710 1, .00 30 .03 6

ATOM 1108 O ARG A 160 17 .398 54 .049 60 .983 1 .00 27 .84 8 ATOM 1109 N GLN A 161 17 .535 55, .017 63 .000 1, .00 26, .77 7

ATOM 1110 NE2 GLN A 161 19, .350 52, .013 67, .864 1. .00 60, .74 7

ATOM llll OE1 GLN A 161 20 .262 53 .735 66 .798 1, .00 59 .57 8

ATOM 1112 CD GLN A 161 19 .355 52 .904 66 .883 1 .00 58 .69 6

ATOM 1113 CG GLN A 161 18 .232 52 .759 65 .891 1 .00 34 .77 6 ATOM 1114 CB GLN A 161 18 .519 53 .945 64 .970 1, .00 30 .48 6

ATOM 1115 CA GLN A 161 17 .801 53 .757 63 .664 1, .00 23 .33 6

ATOM 1116 C GLN A 161 16 .520 52, .971 63 .833 1, .00 29 .67 6

ATOM 1117 O GLN A 161 15, .474 53, .589 63, .955 1. .00 29, .09 8

ATOM 1118 N GLY A 162 16 .517 51 .663 63 .859 1, .00 24 .53 7 ATOM 1119 CA GLY A 162 15 .351 50, .793 64 .031 1, .00 20 .53 6

ATOM 1120 C GLY A 162 15. .104 49. .941 62. .796 1. .00 26, .19 6

ATOM 1121 O GLY A 162 14, .288 50. .249 61, .907 1. .00 22, .33 8

ATOM 1122 N VAL A 165 15 .844 48 .832 62 .774 1, .00 25. .37 7

ATOM 1123 CG2 VAL A 165 18 .242 47, .376 61 .823 1, .00 20 .11 6 ATOM 1124 CGI VAL A 165 16, .767 45, .785 60. .528 1. .00 21 .35 6

ATOM 1125 CB VAL A 165 16, .841 46, .808 61, .703 1. .00 22, .43 6

ATOM 1126 CA VAL A 165 15, .776 47, .891 61, .618 1. ,00 20, .88 6

ATOM 1127 C VAL A 165 14, .383 47. .384 61. .360 1. ,00 24, .44 6

ATOM 1128 O VAL A 165 13, .793 46, .948 62, .359 1. .00 22, .51 8 ATOM 1129 N ASN A 166 13, .847 47, .458 60, .151 1. .00 20, .59 7

ATOM 1130 ND2 ASN A 166 11, ,804 49, .622 59, ,063 1. .00 37, ,01 7

ATOM 1131 ODl ASN A 166 11. .291 48. .862 57. .045 1. ,00 40, .47 8

ATOM 1132 CG ASN A 166 11. .691 48. ,612 58. ,213 1. 00 36, ,75 6

ATOM 1133 CB ASN A 166 12. .084 47. .201 58. ,564 1. ,00 18. ,42 6 ATOM 1134 CA ASN A 166 12. .480 46. .925 60. .012 1. ,00 20. ,41 6

ATOM 1135 C ASN A 166 12. .430 45. ,397 60. .220 1. ,00 27. .19 6

ATOM 1136 O ASN A 166 13. .394 44. ,641 60. .213 1. ,00 20. ,29 8

ATOM 1137 N TYR A 167 11. ,219 44. ,939 60. .323 1. 00 23. ,03 7

ATOM 1138 OH TYR A 167 10. .922 44. .540 66. .485 1. 00 45. ,50 8 ATOM 1139 CD2 TYR A 167 9. .715 44. ,838 63. .205 1. .00 34. .30 6

ATOM 1140 CE2 TYR A 167 10, .084 45, ,141 64. .501 1. ,00 27, .99 6

ATOM 1141 CZ TYR A 167 10. ,625 44. .092 65. .233 1. 00 48. ,07 6

ATOM 1142 CE1 TYR A 167 10. ,871 42. 802 64. ,754 1. 00 30. ,09 6

ATOM 1143 GDI TYR A 167 10. ,582 42. ,588 63. ,401 1. 00 27. .59 6 ATOM 1144 CG TYR A 167 9. ,959 43. ,576 62. ,657 1. 00 30. ,84 6

ATOM 1145 CB TYR A 167 9. ,537 43. ,461 61. ,197 1. 00 25. ,40 6

ATOM 1146 CA TYR A 167 10. ,830 43. 562 60. .383 1. 00 22. .25 6

ATOM 1147 C TYR A 167 10. ,479 43. ,048 58. ,968 1. 00 26. ,82 6

ATOM 1148 O TYR A 167 9. ,785 43. ,740 58. ,230 1. 00 28. ,17 8 ATOM 1149 N PRO A 168 10. ,803 41. ,830 58. ,559 1. 00 24. ,12 7

ATOM 1150 CG PRO A 168 11, ,069 39. ,952 57. .192 1. .00 21. .03 6

ATOM 1151 CD PRO A 168 10. .376 41. ,337 57. .220 1. ,00 18. .66 6

ATOM 1152 CB PRO A 168 11. ,014 39. 509 58. ,639 1. 00 20. ,70 6

ATOM 1153 CA PRO A 168 11. .468 40. ,788 59. .357 1. 00 21. ,08 6 ATOM 1154 C PRO A 168 12. .960 40. ,862 59. .456 1. 00 22. ,02 6

ATOM 1155 O PRO A 168 13. ,492 39. ,981 60. ,180 1. 00 21. ,94 8 ATOM 1156 N ALA A 169 13,.657 41.831 58.841 1..00 15,.90 7

ATOM 1157 CB ALA A 169 15 .736 42 .908 58 .091 1, .00 17, .37 6

ATOM 1158 CA ALA A 169 15 .106 41 .851 58 .949 1, .00 15, .97 6

ATOM 1159 C ALA A 169 15, .607 41 .947 60 .374 1. .00 21, .06 6 ATOM 1160 O ALA A 169 16 .752 41 .565 60 .663 1, .00 21, .07 8

ATOM 1161 N ARG A 170 14 .833 42 .498 61 .289 1 .00 21 .46 7

ATOM 1162 NH2 ARG A 170 13, .387 47, .123 67, .747 1. .00 60. .78 7

ATOM 1163 NH1 ARG A 170 13, .043 47 .610 65 .444 1, .00 49, .63 7

ATOM 1164 CZ ARG A 170 13, .604 46, .896 66, .440 1. .00 59, .33 6 ATOM 1165 NE ARG A 170 14, .377 45 .776 66 .226 1, .00 56, .52 7

ATOM 1166 CD ARG A 170 14 .143 45 .240 64 .921 1, .00 35 .28 6

ATOM 1167 CG ARG A 170 15 .134 44 .173 64 .633 1, .00 26 .86 6

ATOM 1168 CB ARG A 170 14, .382 43 .573 63 .430 1, .00 22, .20 6

ATOM 1169 CA ARG A 170 15 .339 42 .683 62 .653 1, .00 22 .58 6 ATOM 1170 C ARG A 170 15, .423 41 .335 63 .390 1, .00 26, .44 6

ATOM 1171 O ARG A 170 16 .298 41 .175 64 .268 1, .00 24, .76 8

ATOM 1172 N TYR A 171 14, .601 40 .421 63 .006 1, .00 21, .88 7

ATOM 1173 OH TYR A 171 8, .238 39, .587 63, .993 1. .00 30, .27 8

ATOM 1174 CD2 TYR A 171 11, .260 38, .551 62, .366 1. .00 23, .79 6 ATOM 1175 CE2 TYR A 171 9, .930 38 .895 62 .534 1. .00 26, .08 6

ATOM 1176 CZ TYR A 171 9, .544 39, .258 63, .827 1. .00 22, . 66 6

ATOM 1177 CE1 TYR A 171 10 .437 39 .256 64 .849 1, .00 22, .42 6

ATOM 1178 CD1 TYR A 171 11. .754 38. .908 64, .657 1. .00 25, .00 6

ATOM 1179 CG TYR A 171 12 .190 38 .520 63 .397 1, .00 20, .82 6 ATOM 1180 CB TYR A 171 13, .614 38 .157 63, .120 1. .00 20, .76 6

ATOM 1181 CA TYR A 171 14, .662 39, .115 63, .666 1. ,00 21. .12 6

ATOM 1182 C TYR A 171 16 .019 38 .496 63 .429 1. ,00 23, .66 6

ATOM 1183 O TYR A 171 16 .595 38 .612 62 .377 1, .00 19 .53 8

ATOM 1184 N SER A 172 16 .590 37 .805 64, .409 1, .00 21, .25 7 ATOM 1185 OG SER A 172 18, .439 36, .736 66 . .290 1. .00 40, .47 8

ATOM 1186 CB SER A 172 17, .643 36, .002 65 . .430 1, ,00 30. .04 6

ATOM 1187 CA SER A 172 17, .855 37, .114 64. .343 1. ,00 18. .01 6

ATOM 1188 C SER A 172 17, .972 36, .098 63. .241 1, ,00 18. .07 6

ATOM 1189 O SER A 172 19, .076 35. .857 62, .794 1, .00 24, .44 8 ATOM 1190 N GLY A 173 16, .849 35, .497 62, .953 1, .00 20, .49 7

ATOM 1191 CA GLY A 173 16, .895 34 .520 61, .849 1, .00 25, .76 6

ATOM 1192 C GLY A 173 17. ,065 35. .136 60. .466 1. ,00 28. .21 6

ATOM 1193 O GLY A 173 17, ,142 34. .299 59. .561 1. ,00 24. ,65 8

ATOM 1194 N VAL A 174 17, .037 36, .454 60, .298 1. .00 22, .37 7 ATOM 1195 CG2 VAL A 174 14, .544 37, .187 59. .094 1. .00 21. .22 6

ATOM 1196 CGI VAL A 174 15. .868 38. .353 57. .251 1. ,00 16. ,19 6

ATOM 1197 CB VAL A 174 15, .853 37, .860 58. .711 1. ,00 19. .69 6

ATOM 1198 CA VAL A 174 17. .081 37. ,002 58. ,950 1. 00 18. ,39 6

ATOM 1199 C VAL A 174 18. ,302 37. .804 58. ,794 1. ,00 20. ,62 6 ATOM 1200 O VAL A 174 18. ,537 38, .493 59, .767 1. ,00 20. .79 8

ATOM 1201 N MET A 175 19. .071 37. .843 57, ,763 1. ,00 19. ,50 7

ATOM 1202 CE MET A 175 24. .752 37, .650 55, .722 1. ,00 18. ,78 6

ATOM 1203 SD MET A 175 23, .178 36 .899 55, .344 1. .00 28. ,69 16

ATOM 1204 CG MET A 175 22. .276 37. .473 56, .764 1. 00 30. ,99 6 ATOM 1205 CB MET A 175 21. .073 38. .209 56, .458 1. .00 18. .24 6

ATOM 1206 CA MET A 175 20, .269 38. .719 57. .673 1. ,00 19. ,14 6

ATOM 1207 C MET A 175 19. .808 40, .085 57. ,181 1. .00 19. ,60 6

ATOM 1208 O MET A 175 19 .243 40 .124 56, .075 1. .00 19, .68 8

ATOM 1209 N ALA A 176 19, .998 41. .141 57, .911 1. .00 20, .50 7 ATOM 1210 CB ALA A 176 19. .374 43. .342 58. .912 1. 00 17. ,16 6

ATOM 1211 CA ALA A 176 19. .559 42, .523 57. .638 1. ,00 18. ,51 6

ATOM 1212 C ALA A 176 20. .608 43. .146 56. ,758 1. ,00 15. ,58 6

ATOM 1213 O ALA A 176 21. .802 43. .226 57. ,028 1. ,00 18. .20 8

ATOM 1214 N VAL A 177 20 .119 43 .546 55. .592 1. .00 17, .19 7 ATOM 1215 CG2 VAL A 177 20, .837 41. .990 53, .335 1. ,00 16, .29 6

ATOM 1216 CGI VAL A 177 21, .783 44, .025 52. .249 1. ,00 12, .13 6 ATOM 1217 CB VAL A 177 20.739 43.505 53.233 1.00 14.48 6

ATOM 1218 CA VAL A 177 21, .011 44 .188 54 .618 1 .00 18 .22 6

ATOM 1219 C VAL A 177 20 .828 45 .734 54 .489 1 .00 19 .81 6

ATOM 1220 O VAL A 177 19 .728 46 .259 54 .253 1 .00 16 .83 8 ATOM 1221 N ALA A 178 21 .957 46 .444 54 .565 1 .00 17 .27 7

ATOM 1222 CB ALA A 178 23 .054 48 .386 55 .452 1 .00 14 .79 6

ATOM 1223 CA ALA A 178 22 .035 47 .894 54 .418 1 .00 18 .82 6

ATOM 1224 C ALA A 178 22 .445 48 .215 52 .970 1 .00 17 .05 6

ATOM 1225 O ALA A 178 23 .095 47 .447 52 .260 1 .00 16 .34 8 ATOM 1226 N ALA A 179 22 .014 49 .381 52 .483 1 .00 18 .05 7

ATOM 1227 CB ALA A 179 21 .168 50 .710 50 .548 1 .00 14 .34 6

ATOM 1228 CA ALA A 179 22 .317 49 .940 51 .148 1 .00 17 .14 6

ATOM 1229 C ALA A 179 23, .496 50 .901 51 .162 1, .00 16 .16 6

ATOM 1230 O ALA A 179 23 .525 51 .777 52 .044 1 .00 18 .65 8 ATOM 1231 N VAL A 180 24 .451 50 .812 50 .317 1 .00 14 .26 7

ATOM 1232 CG2 VAL A 180 27. .438 49 .981 49 .469 1, .00 17 .76 6

ATOM 1233 CGI VAL A 180 26 .913 50 .487 51 .890 1 .00 16 .51 6

ATOM 1234 CB VAL A 180 26 .964 50 .989 50 .462 1 .00 17 .24 6

ATOM 1235 CA VAL A 180 25, .609 51, .616 50 .075 1, .00 17, .31 6 ATOM 1236 C VAL A 180 25, .586 52, .187 48 .644 1, .00 22, .88 6

ATOM 1237 O VAL A 180 24 .947 51 .671 47 .675 1 .00 20 .50 8

ATOM 1238 N ASP A 181 26 .291 53 .321 48 .446 1 .00 25 .29 7

ATOM 1239 OD2 ASP A 181 27. .098 57 .308 48 .607 1 .00 32 .67 8

ATOM 1240 ODl ASP A 181 28, .547 55 .806 48 .232 1, .00 27 .26 8 ATOM 1241 CG ASP A 181 27. .399 56, .184 48, .028 1. .00 26, .15 6

ATOM 1242 CB ASP A 181 26, .254 55, .570 47 .285 1, .00 24, .05 6

ATOM 1243 CA ASP A 181 26. .408 54, .054 47 .131 1, .00 22, .55 6

ATOM 1244 C ASP A 181 27. .687 53, .624 46, .461 1, .00 28, .00 6

ATOM 1245 ■ O ASP A 181 28 .393 52 .695 46 .923 1, .00 23 .27 8 ATOM 1246 N GLN A 182 28, .038 54 .220 45 .348 1, .00 23 .99 7

ATOM 1247 NE2 GLN A 182 28. .625 56, .347 43, .392 1. .00 59. .18 7

ATOM 1248 OE1 GLN A 182 26. .424 55, .625 43, .700 1, .00 51. .62 8

ATOM 1249 CD GLN A 182 27, .579 55, .498 43 .211 1, .00 57, .87 6

ATOM 1250 CG GLN A 182 28, .188 54, .342 42, .400 1, .00 36, .22 6 ATOM 1251 CB GLN A 182 29, .458 54, .178 43, .178 1. .00 29, .07 6

ATOM 1252 CA GLN A 182 29. .220 53, .827 44, .637 1. .00 20, .63 6

ATOM 1253 C GLN A 182 30. .498 54, .185 45, .347 1. .00 22. .86 6

ATOM 1254 0 GLN A 182 31. .519 53. .726 44, .882 1. .00 27. .70 8

ATOM 1255 N ASN A 183 30. ,450 54, .957 46, .362 1. .00 27. .14 7 ATOM 1256 ND2 ASN A 183 30. ,750 58, .651 46, .572 1. .00 47. .15 7

ATOM 1257 ODl ASN A 183 32. .423 57, .377 45, .640 1. .00 47, .21 8

ATOM 1258 CG ASN A 183 31. .593 57. .633 46, .532 1, .00 47. .14 6

ATOM 1259 CB ASN A 183 31. 436 56. ,677 47. ,691 1. ,00 32. ,89 6

ATOM 1260 CA ASN A 183 31. .656 55. .252 47. .134 1. .00 31. .50 6 ATOM 1261 C ASN A 183 31. ,698 54. .274 48. .330 1. .00 33. .88 6

ATOM 1262 0 ASN A 183 32. ,459 54. .594 49. .245 1. ,00 32. ,01 8

ATOM 1263 N GLY A 184 30. ,838 53. ,306 48. .492 1. ,00 23. ,02 7

ATOM 1264 CA GLY A 184 30. ,887 52. ,499 49. .688 1. ,00 23. ,48 6

ATOM 1265 C GLY A 184 30. ,322 53. ,209 50. ,879 1. ,00 26. ,52 6 ATOM 1266 0 GLY A 184 30. ,461 52. ,723 52. ,013 1. 00 30. ,46 8

ATOM 1267 N GLN A 185 29. 568 54. ,273 50. ,751 1. 00 27. .03 7

ATOM 1268 NE2 GLN A 185 30. ,258 58. .823 51. .467 1. ,00 60. .06 7

ATOM 1269 OE1 GLN A 185 31. ,633 57. .570 53. .078 1. ,00 61. ,27 8

ATOM 1270 CD GLN A 185 30. ,896 57. .806 52. .089 1. ,00 58. ,99 6 ATOM 1271 CG GLN A 185 30. 465 56. .526 51. ,381 1. 00 54. 79 6

ATOM 1272 CB GLN A 185 29. ,023 56. .422 51. ,884 1. ,00 24. ,50 6

ATOM 1273 CA GLN A 185 29. ,012 54. .889 51. ,969 1. 00 24. ,00 6

ATOM 1274 C GLN A 185 27. ,518 54. .587 52. ,026 1. 00 20. ,62 6

ATOM 1275 0 GLN A 185 26. ,870 54. ,488 51. ,012 1. 00 21. ,50 8 ATOM 1276 N ARG A 186 27. .110 54. ,610 53. ,277 1. ,00 20. .28 7

ATOM 1277 NH2 ARG A 186 21. ,131 56. ,372 57. ,060 1. 00 27. .55 7 ATOM 1278 NH1 ARG A 186 22.904 57.626 57.258 .00 35.29 7

ATOM 1279 CZ ARG A 186 22.478 56.400 57.225 .00 38.65 6

ATOM 1280 NE ARG A 186 23.030 55.217 57.167 .00 30.56 7

ATOM 1281 CD ARG A 186 24.081 54.434 56.803 .00 26.76 6 ATOM 1282 CG ARG A 186 24.037 54.280 55.356 .00 19.72 6

ATOM 1283 CB ARG A 186 25.484 54.482 55.024 .00 17.77 6

ATOM 1284 CA ARG A 186 25.719 54.343 53.529 .00 19.83 6

ATOM 1285 C ARG A 186 24.849 55.249 52.697 .00 29.56 6

ATOM 1286 O ARG A 186 25.067 56.444 52.788 .00 26.52 8 ATOM 1287 N ALA A 187 23.822 54.843 52.015 .00 19.87 7

ATOM 1288 CB ALA A 187 22.098 54.655 50.429 .00 22.61 6

ATOM 1289 CA ALA A 187 22.847 55.634 51.325 .00 21.48 6

ATOM 1290 C ALA A 187 22.107 56.312 52.498 .00 23.68 6

ATOM 1291 O ALA A 187 21.762 55.850 53.579 .00 20.22 8 ATOM 1292 N SER A 188 21.706 57.586 52.332 .00 22.67 7

ATOM 1293 OG SER A 188 19.942 59.678 51.654 .00 31.32 8

ATOM 1294 CB SER A 188 20.789 59.773 52.799 .00 27.54 6

ATOM 1295 CA SER A 188 21.069 58.367 53.386 .00 26.75 6

ATOM 1296 C SER A 188 19.792 57.706 53.819 .00 22.34 6 ATOM 1297 O SER A 188 19.413 58.002 54.966 .00 22.84 8

ATOM 1298 N PHE A 189 19.037 56.941 53.001 .00 22.24 7

ATOM 1299 CD2 PHE A 189 17.852 56.033 49.969 .00 17.59 6

ATOM 1300 CE2 PHE A 189 18.514 55.372 48.951 .00 24.96 6

ATOM 1301 CZ PHE A 189 18.791 54.030 49.053 1.00 21.12 6 ATOM 1302 CE1 PHE A 189 18.335 53.422 50.233 1.00 19.91 6

ATOM 1303 CD1 PHE A 189 17.679 54.049 51.248 1..00 19.31 6

ATOM 1304 CG PHE A 189 17.414 55.447 51.104 1.00 24.18 6

ATOM 1305 CB PHE A 189 16.754 56.211 52.243 1.00 17.91 6

ATOM 1306 CA PHE A 189 17.738 56.340 53.411 1.00 19.41 6 ATOM 1307 C PHE A 189 17.900 54.995 54.158 1, .00 13.56 6

ATOM 1308 O PHE A 189 16.915 54.531 54.699 1 00 19.77

ATOM 1309 N SER A 190 19.127 54.513 54.121 1.00 17.30

ATOM 1310 OG SER A 190 20.958 51.491 54.627 1, 00 18.49

ATOM 1311 CB SER A 190 20.614 52.728 54.152 1, 00 19.55 6 ATOM 1312 CA SER A 190 19.310 53.198 54.732 1 00 19.59 6

ATOM 1313 C SER A 190 19.165 53.145 56.233 1, 00 19.58 6

ATOM 1314 O SER A 190 19.993 53.714 56.959 1, 00 22.91 8

ATOM 1315 N THR A 191 18.230 52.366 56.775 1, 00 20.03 7

ATOM 1316 CG2 THR A 191 16.453 50.951 59.871 1, 00 19.20 6 ATOM 1317 OG1 THR A 191 15.685 51.953 57.813 1, 00 23.24 8

ATOM 1318 CB THR A 191 16.775 51.284 58.421 1.00 18.08 6

ATOM 1319 CA THR A 191 17.970 52.140 58.186 1.00 19.69 6

ATOM 1320 C THR A 191 19.214 51.465 58.784 1.00 26.64 6

ATOM 1321 O THR A 191 19.971 50.764 58.083 ,00 20.74 8 ATOM 1322 N TYR A 192 19.509 51.785 60.037 .00 24.10 7

ATOM 1323 OH TYR A 192 20.579 57.242 62.652 .00 42.72 8

ATOM 1324 CD2 TYR A 192 21.008 54.515 60.307 .00 27.03 6

ATOM 1325 CE2 TYR A 192 20.670 55.799 60.760 .00 28.72 6

ATOM 1326 CZ TYR A 192 20.864 56.031 62.103 .00 37.26 6 ATOM 1327 CE1 TYR A 192 21.348 55.083 63.015 .00 36.10 6

ATOM 1328 GDI TYR A 192 21.652 53.820 62.541 .00 25.25 6

ATOM 1329 CG TYR A 192 21.516 53.550 61.169 .00 22.50 6

ATOM 1330 CB TYR A 192 21.910 52.154 60.684 .00 25.18 6

ATOM 1331 CA TYR A 192 20.708 51.213 60.683 .00 17.72 6 ATOM 1332 C TYR A 192 20.258 50.806 62.081 .00 17.55 6

ATOM 1333 O TYR A 192 19.128 50.985 62.559 .00 19.29 8

ATOM 1334 N GLY A 193 21.198 50.136 62.735 .00 19.36 7

ATOM 1335 CA GLY A 193 20.866 49.639 64.090 .00 21.59 6

ATOM 1336 C GLY A 193 21.817 48.449 64.262 .00 23.46 6 ATOM 1337 O GLY A 193 22.550 48.074 63.361 1.00 19.67 8

ATOM 1338 N PRO A 194 21.782 47.949 65.484 1.00 25.90 7 ATOM 1339 CG PRO A 194 20.970 47.337 67.684 .00 27.38 6

ATOM 1340 CD PRO A 194 20.887 48.403 66.615 .00 27.18 6

ATOM 1341 CB PRO A 194 22.239 46.658 67.360 .00 22.45 6

ATOM 1342 CA PRO A 194 22.600 46.837 65.880 .00 28.03 6 ATOM 1343 C PRO A 194 22.412 45.568 65.036 .00 21.43 6

ATOM 1344 O PRO A 194 23.318 44.731 64.998 .00 22.19 8

ATOM 1345 N GLU A 195 21.274 45.405 64.424 .00 20.23 7

ATOM 1346 OE2 GLU A 195 18.569 46.243 65.075 .00 24.87 8

ATOM 1347 OE1 GLU A 195 17.965 44.957 66.720 .00 35.26 8 ATOM 1348 CD GLU A 195 18.409 45.076 65.595 .00 28.08 6

ATOM 1349 CG GLU A 195 18.768 43.825 64.864 .00 21.18 6

ATOM 1350 CB GLU A 195 19.456 43.945 63.541 .00 16.96 6

ATOM 1351 CA GLU A 195 20.940 44.207 63.686 .00 20.96 6

ATOM 1352 C GLU A 195 21.528 44.212 62.285 .00 30.31 6 ATOM 1353 O GLU A 195 21.450 43.118 61.697 .00 23.68 8

ATOM 1354 N ILE A 196 22.053 45.362 61.843 .00 19.60 7

ATOM 1355 CD1 ILE A 196 20.930 47.604 59.167 .00 17.78 6

ATOM 1356 CGI ILE A 196 22.141 47.811 60.018 .00 16.83 6

ATOM 1357 CB ILE A 196 23.248 46.768 60.069 .00 20.89 6 ATOM 1358 CG2 ILE A 196 23.876 46.679 58.658 .00 16.59 6

ATOM 1359 CA ILE A 196 22.643 45.435 60.528 .00 20.36 6

ATOM 1360 C ILE A 196 23.722 44.323 60.503 .00 23.09 6

ATOM 1361 O ILE A 196 24.633 44.261 61.336 .00 20.15 8

ATOM 1362 N GLU A 197 23.649 43.519 59.454 .00 18.90 7 ATOM 1363 OE2 GLU A 197 22.575 38.762 60.250 .00 21.64 8

ATOM 1364 OE1 GLU A 197 24.285 37.564 59.610 1.00 21.92 8

ATOM 1365 CD GLU A 197 23.784 38.629 59.811 1.00 20.50 6

ATOM 1366 CG GLU A 197 24.621 39.884 59.573 . 00 24 . 14 6

ATOM 1367 CB GLU A 197 23.810 41.138 59.266 . 00 19 . 21 6 ATOM 1368 CA GLU A 197 24.642 42.460 59.266 . 00 22 . 15 6

ATOM 1369 C GLU A 197 25.599 42.545 58.109 . 00 18 . 74 6

ATOM 1370 O GLU A 197 26.761 42.130 58.148 . 00 17 . 44 8

ATOM 1371 N ILE A 198 25.090 43.096 56.996 . 00 17 . 50 7

ATOM 1372 CD1 ILE A 198 28.230 41.260 54.456 . 00 17 . 58 6 ATOM 1373 CGI ILE A 198 26.759 41.350 54.022 . 00 15 . 01 6

ATOM 1374 CB ILE A 198 25.746 41.660 55.141 . 00 17 . 39 6

ATOM 1375 CG2 ILE A 198 24.381 41.337 54.553 . 00 14 . 58 6

ATOM 1376 CA ILE A 198 25.916 43.091 55.794 . 00 19 . 95 6

ATOM 1377 C ILE A 198 25.455 44.307 54.934 00 16 . 93 6 ATOM 1378 O ILE A 198 24.294 44.655 55.167 00 18 . 25 8

ATOM 1379 N SER A 199 26.288 44.736 54.001 00 16 . 29 7

ATOM 1380 OG SER A 199 26.677 47.445 54.695 . 00 21 . 30 8

ATOM 1381 CB SER A 199 26.803 47.058 53.330 . 00 20 . 88 6

ATOM 1382 CA SER A 199 25.866 45.811 53.103 . 00 22 . 96 6 ATOM 1383 C SER A 199 26.017 45.418 51.664 . 00 18 . 35 6

ATOM 1384 O SER A 199 26.885 44.606 51.311 . 00 17 .27

ATOM 1385 N ALA A 200 25.292 46.082 50.773 . 00 17 . 99 7

ATOM 1386 CB ALA A 200 24.507 44.703 48.899 . 00 15 . 76 6

ATOM 1387 CA ALA A 200 25.488 45.800 49.315 1. .00 14, ,75 6 ATOM 1388 C ALA A 200 25.057 47.101 48.587 1, .00 18, .96 6

ATOM 1389 O ALA A 200 24.393 47.954 49.211 1, .00 17, .64 8

ATOM 1390 N PRO A 201 25.286 47.223 47.306 1, .00 20, .51 7

ATOM 1391 CG PRO A 201 26.661 47.136 45.380 1, .00 18, .80 6

ATOM 1392 CD PRO A 201 26.109 46.242 46.503 1, .00 16 .13 6 ATOM 1393 CB PRO A 201 25.425 47.930 45.033 1, .00 17, .17 6

ATOM 1394 CA PRO A 201 24.903 48.309 46.424 1. .00 17, ,87 6

ATOM 1395 C PRO A 201 23.380 48.465 46.492 1. .00 19. .20 6

ATOM 1396 O PRO A 201 22.635 47.530 46.248 1, .00 18 .47 8

ATOM 1397 N GLY A 202 22.926 49.697 46.814 1, .00 17 .75 7 ATOM 1398 CA GLY A 202 21.523 49.979 46.902 1, .00 17, .51 6

ATOM 1399 C GLY A 202 21.097 51.274 46.221 1, .00 14, .82 6 ATOM 1400 O GLY A 202 19.959 51.700 46.457 1.00 18.85 8

ATOM 1401 N VAL A 203 21 .915 51 .907 45 .439 1 .00 16 .17 7

ATOM 1402 CG2 VAL A 203 22 .372 54 .486 47 .007 1 .00 17 .72 6

ATOM 1403 CGI VAL A 203 22, .264 55 .632 44 .833 1, .00 25, .37 6 ATOM 1404 CB VAL A 203 22 .496 54 .306 45 .506 1, .00 22 .60 6

ATOM 1405 CA VAL A 203 21 .601 53 .222 44 .828 1, .00 17 .78 6

ATOM 1406 C VAL A 203 21 .846 53 .134 43 .307 1, .00 16 .42 6

ATOM 1407 O VAL A 203 22 .908 52 .700 42 .814 1, .00 17 .23 8

ATOM 1408 N ASN A 204 20 .759 53 .554 42 .619 1, .00 16 .98 7 ATOM 1409 ND2 ASN A 204 22 .049 56 .884 41 .402 1 .00 24 .32 7

ATOM 1410 ODl ASN A 204 19 .987 56 .330 40 .912 1 .00 24 .35 8

ATOM 1411 CG ASN A 204 21 .182 56 .036 40 .900 1, .00 23 .87 6

ATOM 1412 CB ASN A 204 21, .683 54, .651 40. .519 1. .00 18, .76 6

ATOM 1413 CA ASN A 204 20 .810 53 .543 41 .142 1, .00 20 .24 6 ATOM 1414 C ASN A 204 21, .115 52 .155 40. .598 1, .00 19, .44 6

ATOM 1415 O ASN A 204 22, .059 52, .014 39, .793 1. .00 19, .05 8

ATOM 1416 N VAL A 205 20 .304 51 .197 41 .050 1, .00 16 .97 7

ATOM 1417 CG2 VAL A 205 21 .243 49 .080 42 .914 1, .00 19 .48 6

ATOM 1418 CGI VAL A 205 20 .212 47 .386 41 .427 1, .00 16 .95 6 ATOM 1419 CB VAL A 205 20 .268 48 .874 41 .764 1, .00 19 .38 6

ATOM 1420 CA VAL A 205 20, .599 49. .801 40, .597 1. .00 16, .59 6

ATOM 1421 C VAL A 205 19, .701 49, .489 39, .385 1. .00 15 .34 6

ATOM 1422 O VAL A 205 18, .461 49, .433 39, .519 1. .00 16, .76 8

ATOM 1423 N ASN A 206 20, .246 49, .285 38. .208 1. .00 15, .47 7 ATOM 1424 ND2 ASN A 206 18. .309 49, .679 34, .515 1. .00 17 .91 7

ATOM 1425 ODl ASN A 206 20, .019 48, .800 33, .506 1. .00 25, .78 8

ATOM 1426 CG ASN A 206 19 .539 49 .313 34 .549 1. .00 20 .79 6

ATOM 1427 CB ASN A 206 20, .396 49, .386 35, .803 1. .00 18 .19 6

ATOM 1428 CA ASN A 206 19, .494 48, .977 37, .009 1. .00 15. .81 6 ATOM 1429 C ASN A 206 19, .179 47, .474 37, .041 1. .00 18, .98 6

ATOM 1430 O ASN A 206 20, .072 46, .681 37, .314 1. .00 15, .12 8

ATOM 1431 N SER A 207 17, .979 47. .102 36. .724 1. ,00 16, .57 7

ATOM 1432 OG SER A 207 17. .057 43. .866 37. .998 1. ,00 15. .36 8

ATOM 1433 CB SER A 207 17, .276 45, .255 38. .130 1. ,00 20, .35 6 ATOM 1434 CA SER A 207 17, .570 45. .714 36, .707 1. ,00 19, .00 6

ATOM 1435 C SER A 207 16, .343 45, .545 35, .805 1. .00 22, .26 6

ATOM 1436 O SER A 207 15, .858 46, .526 35. .217 1. .00 19, .33 8

ATOM 1437 N THR A 208 15. ,892 44. ,328 35. ,624 1. 00 16, ,17 7

ATOM 1438 CG2 THR A 208 15. .719 41. ,808 33. .875 1. ,00 16, ,30 6 ATOM 1439 OG1 THR A 208 14. .725 41. .939 36. .073 1. ,00 18. .37 8

ATOM 1440 CB THR A 208 14. .653 42. ,429 34. ,738 1. 00 20. ,01 6

ATOM 1441 CA THR A 208 14. .750 43. ,997 34. .777 1. ,00 18, ,19 6

ATOM 1442 C THR A 208 13. .490 44. .637 35. ,310 1. ,00 16. ,06 6

ATOM 1443 O THR A 208 13. .445 44, .830 36. ,515 1. ,00 18, .68 8 ATOM 1444 N TYR A 209 12. .439 44. .866 34. ,532 1. ,00 15^', .66 7

ATOM 1445 OH TYR A 209 8. .061 49. .995 38. .353 1. ,00 24, .32 8

ATOM 1446 CD2 TYR A 209 9. .608 48. .724 35. ,372 1. ,00 21. .41 6

ATOM 1447 CE2 TYR A 209 8. ,725 49. ,455 36. ,172 1. 00 18. .95 6

ATOM 1448 CZ TYR A 209 8. .877 49. .300 37. ,523 1. ,00 21. .61 6 ATOM 1449 CE1 TYR A 209 9. .825 48. .409 38. ,105 1. ,00 20, .67 6

ATOM 1450 CD1 TYR A 209 10. .663 47. ,694 37. ,280 1. .00 16. .67 6

ATOM 1451 CG TYR A 209 10. ,576 47. ,859 35. ,859 1. 00 20. .84 6

ATOM 1452 CB TYR A 209 11. .535 47. .084 34. .944 1. .00 14, .85 6

ATOM 1453 CA TYR A 209 11. .262 45. .514 35. .051 1. ,00 17, .74 6 ATOM 1454 C TYR A 209 10. ,037 45. ,095 34. ,241 1. 00 18. ,77 6

ATOM 1455 0 TYR A 209 10. .306 44. .671 33. .159 1. 00 17. .94 8

ATOM 1456 N THR A 210 8. .808 45. .263 34. ,610 1. 00 17. .67 7

ATOM 1457 CG2 THR A 210 6, .190 44. .160 35. ,943 1. ,00 18, .91 6

ATOM 1458 OG1 THR A 210 6. .688 46. .353 35. ,498 1. 00 22. .54 8 ATOM 1459 CB THR A 210 6. .354 45. .166 34. ,830 1. 00 22. .53 6

ATOM 1460 CA THR A 210 7. .576 44. .936 33. .961 1. ,00 15, .65 6 ATOM 1461 C THR A 210 7.530 45.630 32.615 1.00 21.08 6

ATOM 1462 0 THR A 210 8 .245 46 .596 32 .337 1 .00 21 .96 8

ATOM 1463 N GLY A 211 6 .772 45 .091 31 .686 1 .00 21 .88 7

ATOM 1464 CA GLY A 211 6 .639 45 .433 30 .294 1, .00 16 .41 6 ATOM 1465 C GLY A 211 7 .894 45 .195 29 .496 1 .00 20 .65 6

ATOM 1466 O GLY A 211 8. .073 45 .931 28 .520 1 .00 21 .25 8

ATOM 1467 N ASN A 212 8 .774 44 .261 29 .787 1 .00 18 .14 7

ATOM 1468 ND2 ASN A 212 10, .850 42 .997 25, .498 1, .00 19, .71 7

ATOM 1469 ODl ASN A 212 12 .024 42 .844 27 .473 1, .00 22 .97 8 ATOM 1470 CG ASN A 212 10 .949 43 .075 26 .839 1 .00 24 .04 6

ATOM 1471 CB ASN A 212 9 .727 43 .459 27 .633 1 .00 20 .82 6

ATOM 1472 CA ASN A 212 9 .992 44 .039 29 .021 1 .00 18 .32 6

ATOM 1473 C ASN A 212 10 .824 45 .313 29, .009 1, .00 21 .79 6

ATOM 1474 O ASN A 212 11 .338 45 .759 27, .979 1, .00 19, .34 8 ATOM 1475 N ARG A 213 11 .135 45 .871 30 .138 1, .00 22 .53 7

ATOM 1476 NH2 ARG A 213 9 .855 52 .374 32, .384 1, .00 51, .03 7

ATOM 1477 NH1 ARG A 213 7 .807 51 .315 33 .080 1 .00 51 .09 7

ATOM 1478 CZ ARG A 213 8, .906 51, .367 32. .264 1, .00 58, .97 6

ATOM 1479 NE ARG A 213 9 .162 50 .491 31, .270 1, .00 46, . 66 7 ATOM 1480 CD ARG A 213 8, .664 49 .317 30, .665 1. .00 34, .39 6

ATOM 1481 CG ARG A 213 9 .943 48 .779 30, .110 1, .00 26, .79 6

ATOM 1482 CB ARG A 213 11 .019 48 .187 30, .997 1, ,00 18, .34 6

ATOM 1483 CA ARG A 213 11, .923 47 .139 30, .309 1. .00 21, .06 6

ATOM 1484 C ARG A 213 13 .028 46 .814 31, .301 1, .00 20. .29 6 ATOM 1485 O ARG A 213 13 .179 45 .698 31, .856 1, .00 22, .59 8

ATOM 1486 N TYR A 214 13 .880 47 .783 31 .513 1 .00 19 .32 7

ATOM 1487 OH TYR A 214 16, .343 42, .867 28. .955 1. ,00 22, .20 8

ATOM 1488 CD2 TYR A 214 16 .835 45 .207 31. .687 1, .00 21, .30 6

ATOM 1489 CE2 TYR A 214 16, .827 44, .005 30. .925 1, .00 25, .13 6 ATOM 1490 CZ TYR A 214 16 .378 44 .008 29, .622 1, .00 22 .68 6

ATOM 1491 CE1 TYR A 214 15, .989 45 .194 29, .017 1, .00 22, .65 6

ATOM 1492 CD1 TYR A 214 15, .994 46, .390 29. .760 1. .00 27, .11 6

ATOM 1493 CG TYR A 214 16 .429 46 .385 31, .106 1, .00 20 .60 6

ATOM 1494 CB TYR A 214 16, .401 47 .666 31, .882 1, .00 20, .69 6 ATOM 1495 CA TYR A 214 15 .005 47 .781 32, .462 1, .00 19 .05 6

ATOM 1496 C TYR A 214 14, .859 49, .127 33. .171 1. ,00 27, .90 6

ATOM 1497 O TYR A 214 14, .650 50, .072 32. .408 1. .00 24, .71 8

ATOM 1498 N VAL A 215 14, .933 49, .316 34. .454 1. .00 20, .15 7

ATOM 1499 CG2 VAL A 215 13, .057 52. .183 35. ,930 1. ,00 35. .02 6 ATOM 1500 CGI VAL A 215 12, .963 50, .184 36. .901 1. .00 21, .12 6

ATOM 1501 CB VAL A 215 13, .309 50. .726 35. ,561 1. ,00 22. ,47 6

ATOM 1502 CA VAL A 215 14, .790 50. .566 35. ,197 1. ,00 19. ,28 6

ATOM 1503 C VAL A 215 15, .780 50. .613 36. ,352 1. ,00 26. ,25 6

ATOM 1504 O VAL A 215 16, .115 49, .538 36. ,921 1. ,00 18. ,11 8 ATOM 1505 N SER A 216 16, .242 51, .836 36. .638 1. .00 18, ,21 7

ATOM 1506 OG SER A 216 18, ,922 53. .199 38. .291 1. .00 28, ,38 8

ATOM 1507 CB SER A 216 18. ,437 52, .619 37. ,132 1. ,00 19. .54 6

ATOM 1508 CA SER A 216 17. .173 52. .022 37. ,788 1. ,00 14. ,76 6

ATOM 1509 C SER A 216 16, .379 52, .452 38. .994 1. ,00 15, ,93 6 ATOM 1510 O SER A 216 15, .417 53. .260 38. ,998 1. 00 17. ,76 8

ATOM 1511 N LEU A 217 16. .536 51. .851 40. .157 1. ,00 15. ,32 7

ATOM 1512 CD2 LEU A 217 12. .758 52. .145 40. ,632 1. ,00 14. ,71 6

ATOM 1513 CD1 LEU A 217 12, .750 49, .735 41. .258 1. .00 16, ,65 6

ATOM 1514 CG LEU A 217 13. .614 50. .916 40. ,808 1. ,00 16. .11 6 ATOM 1515 CB LEU A 217 14. .725 51. .092 41. ,795 1. ,00 14. .31 6

ATOM 1516 CA LEU A 217 15. ,935 51. .919 41. 450 1. 00 15. ,26 6

ATOM 1517 C LEU A 217 16. ,939 51. .939 42. ,603 1. ,00 15. .62 6

ATOM 1518 O LEU A 217 18, .064 51, .549 42. .450 1. .00 16, .30 8

ATOM 1519 N SER A 218 16, .586 52, .646 43. .680 1. ,00 20, .18 7 ATOM 1520 OG SER A 218 18, .487 54, .649 44. ,162 1. ,00 18, .87 8

ATOM 1521 CB SER A 218 17. .616 54. .260 45. ,170 1. ,00 14, .18 6 ATOM 1522 CA SER A 218 17.407 52.767 44.891 1.00 15.36 6

ATOM 1523 C SER A 218 16 .603 52 .333 46 .074 1 .00 11 .18 6

ATOM 1524 O SER A 218 15 .384 52 .612 46 .252 1 .00 15 .32 8

ATOM 1525 N GLY A 219 17 .294 51 .647 46 .946 1 .00 13 .73 7 ATOM 1526 CA GLY A 219 16 .541 51 .213 48 .130 1 .00 14 .10 6

ATOM 1527 C GLY A 219 17 .263 50 .056 48 .790 1 .00 13 .62 6

ATOM 1528 O GLY A 219 18 .107 49 .446 48 .142 1 .00 15, .45 8

ATOM 1529 N THR A 220 16 .951 49 .763 50 .039 1 .00 17 .44 7

ATOM 1530 CG2 THR A 220 18 .258 49 .511 52 .936 1 .00 16 .33 6 ATOM 1531 OG1 THR A 220 15 .916 48 .713 52 .516 1 .00 15 .80 8

ATOM 1532 CB THR A 220 17 .286 48 .535 52 .245 1 .00 14 .42 6

ATOM 1533 CA THR A 220 17 .461 48 .580 50 .735 1 .00 17 .50 6

ATOM 1534 C THR A 220 16 .870 47 .306 50 .022 1 .00 19 .97 6

ATOM 1535 O THR A 220 17 .485 46 .256 50, .007 1, .00 15, .81 8 ATOM 1536 N SER A 221 15 .767 47 .403 49 .310 1, .00 20, .24 7

ATOM 1537 OG SER A 221 12 .959 47 .113 48, .642 1 .00 16, .51 8

ATOM 1538 CB SER A 221 13. .930 46 .838 47 .667 1 .00 14 .33 6

ATOM 1539 CA SER A 221 15 .123 46 .390 48 .506 1 .00 12 .90 6

ATOM 1540 C SER A 221 16 .074 46 .003 47 .362 1 .00 15, .12 6 ATOM 1541 O SER A 221 15 .824 44 .880 46, .946 1 .00 17, .53 8

ATOM 1542 N MET A 222 16 .866 46, .875 46, .831 1, .00 17, .58 7

ATOM 1543 CE MET A 222 14 .201 47 .976 44 .365 1 .00 20, .40 6

ATOM 1544 SD MET A 222 15 .531 48, .968 44, .957 1, .00 19, .45 16

ATOM 1545 CG MET A 222 17 .005 48 .396 44 .101 1 .00 13 .71 6 ATOM 1546 CB MET A 222 18, .168 47, .953 44, .968 1, .00 15, .78 6

ATOM 1547 CA MET A 222 17, .828 46. .631 45. .753 1. .00 16, ,66 6

ATOM 1548 C MET A 222 19 .114 46, .047 46, ,344 1, .00 18. .62 6

ATOM 1549 O MET A 222 19 .914 45 .476 45, .641 1 .00 18, .19 8

ATOM 1550 N ALA A 223 19 .567 46, .403 47, .559 1, .00 18, .17 7 ATOM 1551 CB ALA A 223 21 .100 46, .725 49. .390 1, .00 15. .52 6

ATOM 1552 CA ALA A 223 20, .798 45, .907 48. .119 1. .00 17. ,73 6

ATOM 1553 C ALA A 223 20, .550 44. .390 48. .476 1. .00 18. ,17 6

ATOM 1554 O ALA A 223 21 .442 43, .582 48. .237 1. .00 15. .32 8

ATOM 1555 N THR A 224 19, .505 43. .993 49. .096 1. .00 15. .05 7 ATOM 1556 CG2 THR A 224 17, .181 41. .475 50. .592 1. .00 17. ,28 6

ATOM 1557 OG1 THR A 224 17 .567 43. .643 51. .132 1, .00 18, .23 8

ATOM 1558 CB THR A 224 17. .580 42. ,815 49. ,991 1. .00 19. ,73 6

ATOM 1559 CA THR A 224 19, .045 42. ,695 49. .562 1. .00 16. ,32 6

ATOM 1560 C THR A 224 19. .361 41. ,608 48. ,529 1. .00 18. ,10 6 ATOM 1561 O THR A 224 20. .139 40. ,681 48. ,831 1. ,00 18. ,64 8

ATOM 1562 N PRO A 225 18, .887 41. .707 47. .295 1. .00 19. ,05 7

ATOM 1563 CG PRO A 225 18, .136 42. .513 45. .242 1. .00 14. ,92 6

ATOM 1564 CD PRO A 225 17, .891 42. .669 46. ,729 1. ,00 13. ,28 6

ATOM 1565 CB PRO A 225 18. .243 40. ,998 45. ,078 1. ,00 16. ,78 6 ATOM 1566 CA PRO A 225 19. .095 40. ,659 46. ,305 1. ,00 16. ,58 6

ATOM 1567 C PRO A 225 20, ,555 40. .511 46. ,005 1. ,00 18. .27 6

ATOM 1568 O PRO A 225 20, .931 39. .450 45. ,449 1. ,00 18. ,34 8

ATOM 1569 N HIS A 226 21. .430 41. .465 46. .154 1. .00 14. ,72 7

ATOM 1570 CD2 HIS A 226 24. .294 43. ,788 43. .752 1. ,00 18. ,07 6 ATOM 1571 NE2 HIS A 226 23. .748 44. ,863 43. 075 1. 00 19. 52 7

ATOM 1572 CE1 HIS A 226 22. .668 45. ,150 43. ,774 1. .00 14. ,51 6

ATOM 1573 ND1 HIS A 226 22, .493 44. .451 44. ,863 1. .00 17. .68 7

ATOM 1574 CG HIS A 226 23, .536 43. .522 44. ,843 1. .00 17. .79 6

ATOM 1575 CB HIS A 226 23, .792 42. ,501 45. ,921 1. .00 16. .33 6 ATOM 1576 CA HIS A 226 22. .850 41. ,289 45. .803 1. ,00 15. ,23 6

ATOM 1577 C HIS A 226 23. .338 40. ,212 46. ,774 1. ,00 16. ,97 6

ATOM 1578 O HIS A 226 24, .229 39. .428 46. .452 1. ,00 18. ,46 8

ATOM 1579 N VAL A 227 22, .891 40. .288 48. ,000 1. ,00 16. ,78 7

ATOM 1580 CG2 VAL A 227 23. .890 41. ,135 50. ,680 1. ,00 14. ,40 6 ATOM 1581 CGI VAL A 227 23, .403 38. .803 51. .556 1. .00 16. ,55 6

ATOM 1582 CB VAL A 227 23, .078 39. ,851 50. 480 1. ,00 16. ,86 6 ATOM 1583 CA VAL A 227 23.317 39,.328 49,.058 1,.00 19,.40 6

ATOM 1584 C VAL A 227 22, .622 37, .966 48, .813 1. .00 18, .81 6

ATOM 1585 O VAL A 227 23 .389 37 .026 48 .945 1. .00 18 .80 8

ATOM 1586 N ALA A 228 21 .341 37 .929 48 .499 1, .00 16, .53 7 ATOM 1587 CB ALA A 228 19 .234 36, .911 47, .825 1, .00 14, .14 6

ATOM 1588 CA ALA A 228 20 .698 36 .697 48 .134 1 .00 15 .92 6

ATOM 1589 C ALA A 228 21, .468 36, .063 46, .986 1. .00 18. .89 6

ATOM 1590 O ALA A 228 21 .717 34, .844 46, .986 1, .00 18. .57 8

ATOM 1591 N GLY A 229 21 .867 36, .825 45, .976 1, .00 17. .16 7 ATOM 1592 CA GLY A 229 22, .612 36, .385 44, ,821 1. .00 18. .29 6

ATOM 1593 C GLY A 229 23, .921 35, .754 45, .298 1. .00 20. .93 6

ATOM 1594 O GLY A 229 24 .368 34, .727 44, .804 1, .00 18, .60 8

ATOM 1595 N VAL A 230 24 .721 36, .337 46, .178 1, .00 19, .63 7

ATOM 1596 CG2 VAL A 230 27 .344 37, .967 46, .499 1. .00 16. .69 6 ATOM 1597 CGI VAL A 230 28 .071 36 .209 48, .063 1, .00 17, .82 6

ATOM 1598 CB VAL A 230 26 .831 36, .870 47, .428 1, .00 18, .91 6

ATOM 1599 CA VAL A 230 25 .995 35 .834 46 .650 1, .00 21 .31 6

ATOM 1600 C VAL A 230 25, .729 34, .506 47, .398 1. .00 20, .36 6

ATOM 1601 O VAL A 230 26, .608 33. .630 47. .327 1. .00 18, ,14 8 ATOM 1602 N ALA A 231 24 .704 34, .423 48, .186 1, .00 16, ,19 7

ATOM 1603 CB ALA A 231 23 .099 33, .453 49, .852 1. .00 15, ,64 6

ATOM 1604 CA ALA A 231 24, .303 33, .272 48, .943 1. .00 19, .23 6

ATOM 1605 C ALA A 231 24 .106 32, .150 47, .878 1, .00 26, .28 6

ATOM 1606 O ALA A 231 24 .646 31, .063 48, .051 1, .00 18, ,81 8 ATOM 1607 N ALA A 232 23, .425 32, .341 46, .769 1. .00 23, ,31 7

ATOM 1608 CB ALA A 232 22 .170 31, .917 44, .677 1. .00 17, .17 6

ATOM 1609 CA ALA A 232 23 .190 31, .406 45, .678 1. .00 19, .51 6

ATOM 1610 C ALA A 232 24. .513 30, .938 45, .055 1. .00 22, .03 6

ATOM 1611 O ALA A 232 24, .669 29, .709 44. .797 1. .00 21, .60 8 ATOM 1612 N LEU A 233 25, .450 31. .831 44. .890 1. .00 18. ,00 7

ATOM 1613 CD2 LEU A 233 27, .058 32. .978 41. .722 1. ,00 19. ,56 6

ATOM 1614 CD1 LEU A 233 28, .229 34, .741 42. .822 1. ,00 22. .23 6

ATOM 1615 CG LEU A 233 27, ,261 33, .626 43. .063 1. ,00 25. .68 6

ATOM 1616 CB LEU A 233 27, .734 32. .638 44. .100 1. .00 19. ,00 6 ATOM 1617 CA LEU A 233 26 .758 31, .512 44, .380 1. .00 19, ,18 6

ATOM 1618 C LEU A 233 27. .478 30, .583 45. .399 1. .00 32, ,14 6

ATOM 1619 O LEU A 233 28 .163 29 .617 44, .985 1. .00 26, .65 8

ATOM 1620 N VAL A 234 27 .417 30, .811 46, .694 1. .00 23, .95 7

ATOM 1621 CG2 VAL A 234 28, .911 31, .915 49, ,153 1. ,00 20, ,13 6 ATOM 1622 CGI VAL A 234 28, .484 29. .847 50. ,295 1. 00 18. ,16 6

ATOM 1623 CB VAL A 234 28, .054 30. .627 49. .104 1. ,00 20. ,46 6

ATOM 1624 CA VAL A 234 28, .187 30. .033 47. .683 1. ,00 20. ,36 6

ATOM 1625 C VAL A 234 27 .586 28. .631 47. .676 1. ,00 21. ,66 6

ATOM 1626 O VAL A 234 28, .344 27, ,700 47. .665 1. ,00 22. ,83 8 ATOM 1627 N LYS A 235 26, .274 28, ,546 47. .694 1. .00 21, ,98 7

ATOM 1628 NZ LYS A 235 22 .620 23, .743 50, .078 1. .00 29, .40 7

ATOM 1629 CE LYS A 235 22 .462 24, .483 48, .842 1. .00 25, ,24 6

ATOM 1630 CD LYS A 235 23, .510 25, ,553 48. .797 1. .00 30, ,16 6

ATOM 1631 CG LYS A 235 23, .079 26. ,379 47. .585 1. .00 26. .63 6 ATOM 1632 CB LYS A 235 23, .988 27. ,625 47. .594 1. ,00 22. .69 6

ATOM 1633 CA LYS A 235 25, .469 27. .337 47. .688 1. ,00 27. .46 6

ATOM 1634 C LYS A 235 25 .907 26. .501 46, .462 1. .00 34, .52 6

ATOM 1635 O LYS A 235 26 .029 25. .292 46, .590 1. .00 26, .32 8

ATOM 1636 N SER A 236 26, .101 27. .082 45. .314 1. .00 23, .34 7 ATOM 1637 OG SER A 236 27 .255 28, .235 42. .597 1, .00 24, .48 8

ATOM 1638 CB SER A 236 26 .224 27, .305 42, .840 1, .00 20, .87 6

ATOM 1639 CA SER A 236 26 .457 26 .441 44 .069 1, ,00 27 .78 6

ATOM 1640 C SER A 236 27 .893 25 .932 44. .239 1, ,00 32 .81 6

ATOM 1641 O SER A 236 28 .289 24 .881 43. .697 1, ,00 33, .79 8 ATOM 1642 N ARG A 237 28, .779 26, .633 44, .889 1, ,00 27. .94 7

ATOM 1643 NH2 ARG A 237 36 .693 26, .015 46, .199 1. .00 43. .63 7 ATOM 1644 NH1 ARG A 237 34.671 24.734 46.068 1.00 50.62 7

ATOM 1645 CZ ARG A 237 35 .394 25 .866 45 .921 1 .00 55 .63 6

ATOM 1646 NE ARG A 237 34 .768 26 .943 45 .423 1 .00 45 .03 7

ATOM 1647 CD ARG A 237 33 .356 26 .880 44 .981 1 .00 35 .54 6 ATOM 1648 CG ARG A 237 32 .431 27 .220 46 .107 1 .00 36 .78 6

ATOM 1649 CB ARG A 237 31 .048 27 .417 45 .451 1 .00 36 .35 6

ATOM 1650 CA ARG A 237 30 .183 26 .229 45 .057 1 .00 30 .62 6

ATOM 1651 C ARG A 237 30 .294 25 .177 46 .187 1 .00 37 .26 6

ATOM 1652 O ARG A 237 31 .226 24 .364 46 .081 1 .00 32 .10 8 ATOM 1653 N TYR A 238 29 .478 25 .193 47, .202 1 .00 25 .70 7

ATOM 1654 OH TYR A 238 35 .377 26 .896 48 .995 1 .00 38 .64 8

ATOM 1655 CD2 TYR A 238 31 .736 26 .903 49 .223 1 .00 26 .69 6

ATOM 1656 CE2 TYR A 238 33 .029 27 .369 49 .095 1 .00 30 .27 6

ATOM 1657 CZ TYR A 238 34 .086 26 .481 49 .141 1 .00 38 .00 6 ATOM 1658 CE1 TYR A 238 33 .828 25 .135 49 .328 1 .00 33 .49 6

ATOM 1659 CD1 TYR A 238 32 .531 24, .676 49, .487 1, .00 30, .85 6

ATOM 1660 CG TYR A 238 31 .457 25 .546 49 .441 1 .00 33 .19 6

ATOM 1661 CB TYR A 238 30 .081 24. .961 49, .606 1, .00 24, .64 6

ATOM 1662 CA TYR A 238 29 .529 24 .325 48 .331 1 .00 23 .05 6 ATOM 1663 C TYR A 238 28 .122 23 .867 48 .656 1 .00 25 .25 6

ATOM 1664 O TYR A 238 27 .514 24 .266 49, .659 1, .00 30 .61 8

ATOM 1665 N PRO A 239 27 .688 22 .920 47 .848 1 .00 27 .20 7

ATOM 1666 CG PRO A 239 27, .396 21, .618 45, ,894 1, ,00 27, ,10 6

ATOM 1667 CD PRO A 239 28 .420 22. .386 46, .677 1, .00 28, .97 6 ATOM 1668 CB PRO A 239 26, .237 21, .401 46, .789 1, .00 27, .07 6

ATOM 1669 CA PRO A 239 26, .374 22, .336 47. .936 1. .00 24. .32 6

ATOM 1670 C PRO A 239 26 .018 21, .775 49. .271 1, .00 27, .11 6

ATOM 1671 O PRO A 239 24 .832 21 .805 49, .646 1. .00 34 .83 8

ATOM 1672 N SER A 240 27 .032 21, .338 49, .983 1, .00 28, .71 7 ATOM 1673 OG SER A 240 28, .905 20, .696 51, ,933 1, .00 44, .71 8

ATOM 1674 CB SER A 240 27, .802 19. .807 51, ,651 1, .00 32. .98 6

ATOM 1675 CA SER A 240 26, .658 20. .772 51. ,295 1. .00 31. .35 6

ATOM 1676 C SER A 240 26, .514 21, .852 52, ,339 1, .00 35. .23 6

ATOM 1677 O SER A 240 26, .021 21, .373 53, ,361 1. .00 33. .72 8 ATOM 1678 N TYR A 241 26, .917 23. .099 52, ,126 1. .00 33. .49 7

ATOM 1679 OH TYR A 241 32, .514 26. .940 53, .424 1, .00 39. .30 8

ATOM 1680 CD2 TYR A 241 28, .974 26. .952 52. .686 1. .00 28, .15 6

ATOM 1681 CE2 TYR A 241 30, .301 27. .321 52. ,920 1. .00 31, .52 6

ATOM 1682 CZ TYR A 241 31. .258 26. .429 53. ,256 1. .00 27, ,23 6 ATOM 1683 CE1 TYR A 241 30. ,883 25. .121 53. ,346 1. .00 31. ,25 6

ATOM 1684 CD1 TYR A 241 29. .567 24. .730 53. ,129 1. .00 37. .65 6

ATOM 1685 CG TYR A 241 28. .574 25. ,641 52. ,769 1. ,00 33. .89 6

ATOM 1686 CB TYR A 241 27. .141 25. .321 52. .486 1. .00 29. .46 6

ATOM 1687 CA TYR A 241 26, .737 24. .060 53. .228 1. .00 26. .63 6 ATOM 1688 C TYR A 241 25. .346 24. ,320 53. 736 1. 00 25. ,38 6

ATOM 1689 O TYR A 241 24. .430 24. ,339 52. ,874 1. ,00 29. .17 8

ATOM 1690 N THR A 242 25. .146 24. .489 55. .044 1. ,00 24. ,62 7

ATOM 1691 CG2 THR A 242 23. .731 22. ,950 57. .120 1. ,00 42. ,00 6

ATOM 1692 OG1 THR A 242 24. .567 25. ,108 57. ,591 1. ,00 31. ,68 8 ATOM 1693 CB THR A 242 23. .519 24. ,442 56. ,951 1. 00 33. .79 6

ATOM 1694 CA THR A 242 23. .802 24. ,846 55. ,488 1. ,00 26. ,63 6

ATOM 1695 C THR A 242 23. .625 26. ,399 55. ,366 1. ,00 31. ,00 6

ATOM 1696 O THR A 242 24. .567 27. ,112 55. ,026 1. ,00 24. ,59 8

ATOM 1697 N ASN A 243 22. ,455 26. ,868 55. 672 1. 00 23. ,08 7 ATOM 1698 ND2 ASN A 243 19. ,284 28. ,150 58. ,303 1. ,00 25. ,05 7

ATOM 1699 ODl ASN A 243 21. .147 26. ,869 58. ,079 1. ,00 29. ,80 8

ATOM 1700 CG ASN A 243 20, .365 27. .718 57. ,665 1. ,00 31. .10 6

ATOM 1701 CB ASN A 243 20. .705 28. .307 56. ,289 1. ,00 26. .51 6

ATOM 1702 CA ASN A 243 22. ,106 28. 250 55. 754 1. 00 21. 33 6 ATOM 1703 C ASN A 243 23. .148 28. ,899 56. ,695 1. ,00 27. .65 6

ATOM 1704 O ASN A 243 23. .802 29. ,886 56. 362 1. 00 25. ,83 8 ATOM 1705 N ASN A 244 23.468 28.330 57.867 1.00 26.03 7

ATOM 1706 ND2 ASN A 244 22 .587 29 .649 60 .811 1 .00 34 .79 7

ATOM 1707 ODl ASN A 244 22 .802 27 .357 61 .068 1 .00 44 .84 8

ATOM 1708 CG ASN A 244 23 .191 28 .490 60 .756 1 .00 35 .31 6 ATOM 1709 CB ASN A 244 24 .543 28 .310 60 .131 1 .00 23 .50 6

ATOM 1710 CA ASN A 244 24 .468 28 .913 58 .741 1 .00 23 .28 6

ATOM 1711 C ASN A 244 25 .852 29 .042 58 .177 1 .00 20 .60 6

ATOM 1712 O ASN A 244 26 .588 29 .986 58 .528 1, .00 25 .11 8

ATOM 1713 N GLN A 245 26 .288 28 .065 57 .405 1, .00 26 .52 7 ATOM 1714 NE2 GLN A 245 29, .731 23 .917 56, .910 1, .00 46, .58 7

ATOM 1715 OE1 GLN A 245 27, .592 23 .848 56, .288 1, .00 31, .92 8

ATOM 1716 CD GLN A 245 28 .495 24 .413 56 .857 1 .00 31 .91 6

ATOM 1717 CG GLN A 245 28 .158 25 .769 57 .424 1, .00 31 .13 6

ATOM 1718 CB GLN A 245 28 .079 26 .789 56 .257 1, .00 22 .63 6 ATOM 1719 CA GLN A 245 27, .641 28 .159 56 .822 1, .00 25 .01 6

ATOM 1720 C GLN A 245 27 .700 29 .217 55 .724 1 .00 22 .64 6

ATOM 1721 O GLN A 245 28 .812 29 .747 55 .628 1 .00 22 .33 8

ATOM 1722 N ILE A 246 26, .579 29, .386 55, .029 1. .00 19, .17 7

ATOM 1723 CD1 ILE A 246 24. .121 28, .540 51. .765 1. .00 24, .28 6 ATOM 1724 CGI ILE A 246 25. .491 28, .913 52. .305 1. .00 22, .92 6

ATOM 1725 CB ILE A 246 25, .388 30. .250 53, .066 1, .00 24 .08 6

ATOM 1726 CG2 ILE A 246 25, .359 31, .365 52, .019 1. .00 15, .42 6

ATOM 1727 CA ILE A 246 26, .626 30, .376 53, .946 1, .00 20, .73 6

ATOM 1728 C ILE A 246 26, .625 31, .770 54, .595 1. .00 21, .00 6 ATOM 1729 O ILE A 246 27, .450 32 .600 54, .231 1, .00 21 .98 8

ATOM 1730 N ARG A 247 25, .815 31, .946 55, .595 1. .00 17, .95 7

ATOM 1731 NH2 ARG A 247 21, .172 36, .496 61, ,002 1. .00 23, ,24 7

ATOM 1732 NH1 ARG A 247 20. .813 34, .285 60, .509 1. .00 25, .64 7

ATOM 1733 CZ ARG A 247 21. .541 35, .380 60. .384 1. .00 22. .42 6 ATOM 1734 NE ARG A 247 22. .621 35, .221 59. .659 1. .00 20. .46 7

ATOM 1735 CD ARG A 247 23. .075 33. .985 59. .041 1. ,00 23. .20 6

ATOM 1736 CG ARG A 247 24. .278 34. .245 58. .197 1. ,00 24. .44 6

ATOM 1737 CB ARG A 247 24. ,599 32. .992 57. .408 1. ,00 19. ,57 6

ATOM 1738 CA ARG A 247 25. .664 33. ,174 56. .359 1. ,00 19. .19 6 ATOM 1739 C ARG A 247 27. .002 33. .597 56. .927 1. .00 25. .09 6

ATOM 1740 O ARG A 247 27. .519 34. .707 56. .756 1. ,00 22. ,02 8

ATOM 1741 N GLN A 248 27. .650 32. .632 57. .527 1. ,00 20. ,56 7

ATOM 1742 NE2 GLN A 248 31, .226 29, .317 59, .418 1. .00 42, ,74 7

ATOM 1743 OE1 GLN A 248 30. .871 30, .465 61. .389 1. ,00 46, ,93 8 ATOM 1744 CD GLN A 248 30. ,990 30, .383 60. .165 1. ,00 51, ,60 6

ATOM 1745 CG GLN A 248 30. .736 31, .700 59. .458 1. .00 35. .08 6

ATOM 1746 CB GLN A 248 29. .288 31. .684 59. .012 1. .00 25. .69 6

ATOM 1747 CA GLN A 248 28. ,981 32. .908 58. .114 1. .00 22. .60 6

ATOM 1748 C GLN A 248 30. ,017 33. .161 57. ,069 1. ,00 21. .32 6 ATOM 1749 O GLN A 248 30. ,901 33. .970 57. ,349 1. .00 21. .82 8

ATOM 1750 N ARG A 249 29. ,967 32. .465 55. .934 1. .00 19, .01 7

ATOM 1751 NH2 ARG A 249 35. .824 29. .779 51. .206 1. ,00 40. .53 7

ATOM 1752 NH1 ARG A 249 34. .826 29. .545 53. .357 1. .00 37. .14 7

ATOM 1753 CZ ARG A 249 34. ,844 29. .899 52. ,078 1. ,00 40. .51 6 ATOM 1754 NE ARG A 249 33. ,791 30. .659 51. ,779 1. .00 39. .30 7

ATOM 1755 CD ARG A 249 33. ,221 31. .268 52. ,999 1. ,00 30. ,94 6

ATOM 1756 CG ARG A 249 31. ,842 31. .741 52. ,706 1. ,00 25. ,20 6

ATOM 1757 CB ARG A 249 30. .911 31. .639 53. ,899 1. ,00 22. ,51 6

ATOM 1758 CA ARG A 249 31. .014 32. .723 54. ,960 1. ,00 22. ,86 6 ATOM 1759 C ARG A 249 30. .910 34, .140 54. .373 1. ,00 17. ,79 6

ATOM 1760 O ARG A 249 31. .962 34. .746 54. .144 1. ,00 19. ,55 8

ATOM 1761 N ILE A 250 29. .677 34. .545 54. .142 1. ,00 18. .51 7

ATOM 1762 CD1 ILE A 250 26. .140 35, .220 51. .913 1. ,00 18. .42 6

ATOM 1763 CGI ILE A 250 27, .639 35, .352 52. .018 1. .00 18. .91 6 ATOM 1764 CB ILE A 250 27, ,933 36. .102 53. .316 1. 00 23. .88 6

ATOM 1765 CG2 ILE A 250 27. ,571 37. .586 53. ,219 1. 00 20. ,31 6 ATOM 1766 CA ILE A 250 29.420 35.892 53.596 00 22.42 6

ATOM 1767 C ILE A 250 29.936 36.914 54.662 00 20.70 6

ATOM 1768 O ILE A 250 30.697 37.770 54.268 00 20.41 8

ATOM 1769 N ASN A 251 29.611 36.778 55.909 00 16.56 7 ATOM 1770 ND2 ASN A 251 27.085 37.213 59.132 00 21.27 7

ATOM 1771 ODl ASN A 251 27.518 38.556 57.396 00 20.03 8

ATOM 1772 CG ASN A 251 27.884 37.722 58.234 00 20.12 6

ATOM 1773 CB ASN A 251 29.340 37.365 58.291 1.00 14.58 6

ATOM 1774 CA ASN A 251 30.053 37.650 56.988 00 19.60 6 ATOM 1775 C ASN A 251 31.548 37.703 57.148 00 22.53 6

ATOM 1776 O ASN A 251 32.201 38.759 57.273 00 20.64 8

ATOM 1777 N GLN A 252 32.182 36.536 57.064 00 22.96 7

ATOM 1778 NE2 GLN A 252 33.954 32.143 57.495 00 26.44 7

ATOM 1779 OE1 GLN A 252 34.257 32.144 59.601 00 34.65 8 ATOM 1780 CD GLN A 252 33.983 32.794 58.626 00 33.52 6

ATOM 1781 CG GLN A 252 33.666 34.300 58.676 00 32.35 6

ATOM 1782 CB GLN A 252 34.161 35.012 57.438 00 19.97 6

ATOM 1783 CA GLN A 252 33.609 36.444 57.294 00 22.53 6

ATOM 1784 C GLN A 252 34.428 37.094 56.208 00 21.65 6 ATOM 1785 O GLN A 252 35.605 37.391 56.464 00 22.97 8

ATOM 1786 N THR A 253 33.896 37.119 55.011 00 18.20 7

ATOM 1787 CG2 THR A 253 35.122 35.288 53.155 00 26.77 6

ATOM 1788 OG1 THR A 253 33.244 36.457 52.335 00 20.36 8

ATOM 1789 CB THR A 253 34.578 36.633 52.726 00 22.10 6 ATOM 1790 CA THR A 253 34.689 37.647 53.898 00 19.73 6

ATOM 1791 C THR A 253 34.340 39.071 53.463 00 19.95 6

ATOM 1792 O THR A 253 34.913 39.503 52.482 00 20.25 8

ATOM 1793 N ALA A 254 33.429 39.726 54.117 00 19.64 7

ATOM 1794 CB ALA A 254 31.740 41.369 54.617 00 18.36 6 ATOM 1795 CA ALA A 254 32.987 41.091 53.782 00 24.84 6

ATOM 1796 C ALA A 254 34.120 42.087 53.921 00 21.63 6

ATOM 1797 O ALA A 254 35.058 41.906 54.708 00 20.05 8

ATOM 1798 N THR A 255 34.176 43.140 53.147 00 21.26 7

ATOM 1799 CG2 THR A 255 36.230 46.013 52.142 00 25.89 6 ATOM 1800 OG1 THR A 255 35.698 44.059 51.035 00 26.17 8

ATOM 1801 CB THR A 255 35.139 44.994 51.925 00 22.57 6

ATOM 1802 CA THR A 255 35.193 44.192 53.240 00 21.57 6

ATOM 1803 C THR A 255 34.718 45.197 54.248 00 19.15 6

ATOM 1804 O THR A 255 33.550 45.592 54.161 00 19.14 8 ATOM 1805 N TYR A 256 35.458 45.555 55.262 00 21.54 7

ATOM 1806 OH TYR A 256 35.344 50.333 61.399 00 27.67 8

ATOM 1807 CD2 TYR A 256 35.133 47.291 59.487 00 18.22 6

ATOM 1808 CE2 TYR A 256 34.941 48.186 60.527 00 19.33 6

ATOM 1809 CZ TYR A 256 35.581 49.413 60.435 00 23.39 6 ATOM 1810 CE1 TYR A 256 36.360 49.758 59.359 00 21.50 6

ATOM 1811 CD1 TYR A 256 36.542 48.786 58.359 00 24.73 6

ATOM 1812 CG TYR A 256 35.930 47.528 58.414 00 18.54 6

ATOM 1813 CB TYR A 256 36.204 46.511 57.365 00 19.73 6

ATOM 1814 CA TYR A 256 35.044 46.469 56.350 00 22.62 6 ATOM 1815 C TYR A 256 34.821 47.867 55 744 00 21.73 6

ATOM 1816 O TYR A 256 35.663 48.297 54.920 00 21.87 8

ATOM 1817 N LEU A 257 33.684 48.448 56.082 00 19.62 7

ATOM 1818 CD2 LEU A 257 32.720 49.475 52.464 00 18.99 6

ATOM 1819 CD1 LEU A 257 30.367 48.966 53.151 00 21.76 6 ATOM 1820 CG LEU A 257 31.817 48.960 53.516 00 20.48 6

ATOM 1821 CB LEU A 257 31.922 49.666 54.836 00 19.04 6

ATOM 1822 CA LEU A 257 33.313 49.753 55.519 00 27.37 6

ATOM 1823 C LEU A 257 33.263 50.866 56.576 00 27.50 6

ATOM 1824 O LEU A 257 33.107 52.015 56.207 00 25.78 ATOM 1825 N GLY A 258 33.152 50.534 57.828 00 22.89

ATOM 1826 CA GLY A 258 33.057 51.513 58.894 00 21.99 ATOM 1827 C GLY A 258 32.163 50.880 59.937 1.00 24.86 6

ATOM 1828 O GLY A 258 31 .926 49 .672 60 .084 1 .00 24 .18 8

ATOM 1829 N SER A 259 31 .569 51 .743 60 .724 1 .00 20 .88 7

ATOM 1830 OG SER A 259 29, .158 52 .213 63 .426 1, .00 31 .61 8 ATOM 1831 CB SER A 259 29 .974 52 .583 62 .307 1 .00 24 .36 6

ATOM 1832 CA SER A 259 30, .733 51 .337 61 .822 1 .00 24 .45 6

ATOM 1833 C SER A 259 29, .770 50, .171 61, .540 1, .00 27, .60 6

ATOM 1834 O SER A 259 28, .843 50 .318 60 .730 1, .00 22 .13 8

ATOM 1835 N PRO A 260 29 .842 49 .141 62 .343 1 .00 21 .74 7 ATOM 1836 CG PRO A 260 31 .036 47 .393 63 .408 1 .00 24 .82 6

ATOM 1837 CD PRO A 260 30, .994 48 .911 63 .310 1 .00 25 .73 6

ATOM 1838 CB PRO A 260 29 .514 47 .117 63 .404 1 .00 21 .61 6

ATOM 1839 CA PRO A 260 29, .031 47 .947 62 .217 1, .00 19 .23 6

ATOM 1840 C PRO A 260 27 .609 48 .328 62 .386 1 .00 21 .40 6 ATOM 1841 O PRO A 260 26, .757 47 .607 61 .855 1 .00 21 .68 8

ATOM 1842 N SER A 261 27, .313 49, .416 63, .117 1, .00 24, .57 7

ATOM 1843 OG SER A 261 26, .184 51 .724 64. .185 1 .00 39 .92 8

ATOM 1844 CB SER A 261 25 .584 50 .471 64 .588 1 .00 26 .32 6

ATOM 1845 CA SER A 261 25, .846 49, .736 63. .266 1. .00 21, .73 6 ATOM 1846 C SER A 261 25. .265 50, .281 61, .945 1, .00 22, .12 6

ATOM 1847 O SER A 261 24. .035 50, .276 61, .642 1, .00 24, .01 8

ATOM 1848 N LEU A 262 26. .160 50 .717 61, .066 1 .00 16 .86 7

ATOM 1849 CD2 LEU A 262 25 .190 53 .985 60 .792 1 .00 19 .12 6

ATOM 1850 CD1 LEU A 262 27. .301 54 .691 59. .591 1, .00 24 .54 6 ATOM 1851 CG LEU A 262 26. .558 53, .641 60, .336 1, .00 21, .01 6

ATOM 1852 CB LEU A 262 26, ,462 52 .472 59, .338 1, .00 17 .76 6

ATOM 1853 CA LEU A 262 25, .690 51 .214 59 .777 1 .00 20 .61 6

ATOM 1854 C LEU A 262 25, ,743 50, .137 58, .665 1, .00 22, .18 6

ATOM 1855 O LEU A 262 24. ,898 50, .044 57. .784 1. .00 20, .95 8 ATOM 1856 N TYR A 263 26, ,839 49, .424 58. .640 1, .00 20, .86 7

ATOM 1857 OH TYR A 263 29. .102 54, .204 55. .461 1. .00 26, .47 8

ATOM 1858 CD2 TYR A 263 29, .566 51, .211 57, .467 1, .00 26, .53 6

ATOM 1859 CE2 TYR A 263 29, .687 52 .535 57, .088 1. .00 20, .10 6

ATOM 1860 CZ TYR A 263 28. .983 52, .914 55, .953 1, .00 29, .82 6 ATOM 1861 CE1 TYR A 263 28. .242 51, .962 55, .229 1, .00 23, .04 6

ATOM 1862 CD1 TYR A 263 28. .099 50 .658 55, .660 1. .00 21 .97 6

ATOM 1863 CG TYR A 263 28. .770 50, .273 56, ,804 1, .00 22, .44 6

ATOM 1864 CB TYR A 263 28. .675 48. .901 57. ,334 1. .00 18, .72 6

ATOM 1865 CA TYR A 263 27. .257 48, .431 57, ,689 1. .00 19, .34 6 ATOM 1866 C TYR A 263 27. ,356 46, .941 58. .112 1. .00 20, .55 6

ATOM 1867 O TYR A 263 27. .557 46, .151 57. .208 1, .00 20, .61 8

ATOM 1868 N GLY A 264 27. .252 46, .559 59, .371 1. .00 23, .63 7

ATOM 1869 CA GLY A 264 27. ,399 45. ,182 59. .846 1. ,00 23. .19 6

ATOM 1870 C GLY A 264 28. ,879 44. ,821 59, .611 1. ,00 20. .89 6 ATOM 1871 O GLY A 264 29. ,792 45. ,612 59. .912 1. .00 22, .16 8

ATOM 1872 N ASN A 265 29. .016 43, .657 58, .986 1, .00 20, .54 7

ATOM 1873 ND2 ASN A 265 28, .705 40 .460 59, .762 1. .00 18, .28 7

ATOM 1874 ODl ASN A 265 31. .001 40, .510 60, .158 1. ,00 22, .08 8

ATOM 1875 CG ASN A 265 29. .953 40, .799 59, .474 1. .00 23. .12 6 ATOM 1876 CB ASN A 265 30. .177 41, .671 58. ,249 1. .00 22, .83 6

ATOM 1877 CA ASN A 265 30. .354 43, .162 58. .629 1. .00 18, ,37 6

ATOM 1878 C ASN A 265 30. .933 43. .918 57. ,463 1. ,00 17. ,82 6

ATOM 1879 O ASN A 265 32. ,101 43, .734 57. ,184 1. .00 19. ,89 8

ATOM 1880 N GLY A 266 30. .149 44, .653 56. .673 1. .00 18. .61 7 ATOM 1881 CA GLY A 266 30. .810 45. .365 55. .570 1. .00 16. .52 6

ATOM 1882 C GLY A 266 30. .147 44, .955 54. .258 1. ,00 14, .39 6

ATOM 1883 O GLY A 266 29. .012 44, .489 54. .261 1. .00 17. .41 8

ATOM 1884 N LEU A 267 30. .938 45, .180 53. .248 1. ,00 17, .00 7

ATOM 1885 CD2 LEU A 267 31. .818 46, .464 48. .528 1. .00 20, .10 6 ATOM 1886 CD1 LEU A 267 29. .447 46 .337 49, .267 1, .00 17 .92 6

ATOM 1887 CG LEU A 267 30. .836 45, .825 49, .468 1, .00 21. .20 6 ATOM 1888 CB LEU A 267 31.195 45.897 50.957 1.00 17.31 6

ATOM 1889 CA LEU A 267 30 .473 44 .933 51 .911 1 .00 19 .77 6

ATOM 1890 C LEU A 267 30 .613 43 .483 51 .457 1 .00 19 .17 6

ATOM 1891 O LEU A 267 31 .713 43 .027 51 .499 1 .00 18 .24 8 ATOM 1892 N VAL A 268 29 .515 42 .890 51 .007 1 .00 18 .79 7

ATOM 1893 CG2 VAL A 268 28 .108 39 .597 49 .871 1 .00 21 .18 6

ATOM 1894 CGI VAL A 268 27 .562 41 .922 48 .977 1 .00 17 .72 6

ATOM 1895 CB VAL A 268 28. .154 41, .080 50 .102 1 .00 18 .91 6

ATOM 1896 CA VAL A 268 29 .593 41 .487 50 .497 1 .00 19 .33 6 ATOM 1897 C VAL A 268 30 .656 41 .429 49 .439 1 .00 21 .97 6

ATOM 1898 O VAL A 268 30 .848 42 .376 48 .631 1 .00 22 .07 8

ATOM 1899 N HIS A 269 31 .459 40 .358 49 .345 1 .00 18 .59 7

ATOM 1900 CD2 HIS A 269 36 .030 41 .419 48 .127 1 .00 21 .57 6

ATOM 1901 NE2 HIS A 269 36 .783 41 .004 47 .080 1 .00 22 .77 7 ATOM 1902 CE1 HIS A 269 36 .264 39 .941 46 .468 1 .00 21 .29 6

ATOM 1903 ND1 HIS A 269 35 .180 39 .655 47 .082 1 .00 20 .84 7

ATOM 1904 CG HIS A 269 35 .025 40 .514 48 .132 1 .00 18 .43 6

ATOM 1905 CB HIS A 269 33 .878 40 .370 49 .071 1 .00 17 . 66 6

ATOM 1906 CA HIS A 269 32, .544 40, .254 48, .361 1, .00 19, .72 6 ATOM 1907 C HIS A 269 32 .331 38, .894 47, .726 1, .00 22 .36 6

ATOM 1908 O HIS A 269 32, .629 37. .899 48, .397 1. .00 20, .96 8

ATOM 1909 N ALA A 270 31 .766 38 .818 46 .559 1 .00 22 .28 7

ATOM 1910 CB ALA A 270 30 .573 37 .918 44 .601 1 .00 17 .20 6

ATOM 1911 CA ALA A 270 31 .431 37 .593 45 .842 1 .00 21 .32 6 ATOM 1912 C ALA A 270 32, .677 36, .745 45, .532 1, .00 26 .89 6

ATOM 1913 O ALA A 270 32, .564 35, .516 45, .514 1, .00 23 .48 8

ATOM 1914 N GLY A 271 33 .851 37 .281 45. .257 1, .00 20 .68 7

ATOM 1915 CA GLY A 271 35, .107 36, .638 44, .880 1, .00 24 .34 6

ATOM 1916 C GLY A 271 35, .612 35, .980 46, .150 1, .00 30 .38 6 ATOM 1917 O GLY A 271 35 .866 3 .786 46 .145 1. .00 29 .87 8

ATOM 1918 N ARG A 272 35, .718 36, .672 47, .271 1, .00 25, .63 7

ATOM 1919 NH2 ARG A 272 39, .216 41, .988 51, .543 1, .00 39 .62 7

ATOM 1920 NH1 ARG A 272 37 .245 41, .084 52, .031 1, .00 33 .73 7

ATOM 1921 CZ ARG A 272 38, .322 41, .035 51, .261 1. .00 29, .01 6 ATOM 1922 NE ARG A 272 38, .462 40, .006 50, .408 1, .00 27, .85 7

ATOM 1923 CD ARG A 272 37. .427 38 .979 50, .545 1, .00 24 .30 6

ATOM 1924 CG ARG A 272 37. ,529 37. ,929 49. ,449 1. ,00 24, .96 6

ATOM 1925 CB ARG A 272 36. .387 36. ,959 49. ,653 1. ,00 24, .60 6

ATOM 1926 CA ARG A 272 36. .154 35. ,998 48. .480 1. ,00 24, .91 6 ATOM 1927 C ARG A 272 35. .202 34, ,911 48. .922 1. .00 26, .40 6

ATOM 1928 O ARG A 272 35, .641 33, .851 49. .431 1. .00 28. .24 8

ATOM 1929 N ALA A 273 33, .914 35, .188 48, .929 1, .00 19 .69 7

ATOM 1930 CB ALA A 273 31, .517 34, .902 49, .536 1. .00 20. .87 6

ATOM 1931 CA ALA A 273 32. .936 34. .244 49. ,474 1. ,00 24, .30 6 ATOM 1932 C ALA A 273 32. .968 32. .852 48. ,766 1. ,00 27, .05 6

ATOM 1933 O ALA A 273 32. .536 31, .854 49. ,362 1. ,00 24, .22 8

ATOM 1934 N THR A 274 33. .319 32, .767 47. ,501 1. .00 24, .53 7

ATOM 1935 CG2 THR A 274 31. .085 32, .479 45. ,548 1. ,00 21, .97 6

ATOM 1936 OG1 THR A 274 33. .334 32, .912 44. .673 1. .00 21, .52 8 ATOM 1937 CB THR A 274 32. ,493 32, .003 45. .307 1. ,00 23, .92 6

ATOM 1938 CA THR A 274 33. .266 31. .637 46. .614 1. ,00 27, .06 6

ATOM 1939 C THR A 274 34. .616 30, .968 46. .450 1. ,00 23, .60 6

ATOM 1940 O THR A 274 34. .742 30. .024 45. .712 1. ,00 26. .35 8

ATOM 1941 N GLN A 275 35. .613 31. .466 47. .075 1. ,00 24, .38 7 ATOM 1942 NE2 GLN A 275 38. .108 33. .169 50. .922 1. .00 25. .89 7

ATOM 1943 OE1 GLN A 275 39. .935 31. .618 50. .540 1. .00 44. .50 8

ATOM 1944 CD GLN A 275 38. .904 32. .283 50. ,229 1. 00 56. .87 6

ATOM 1945 CG GLN A 275 38. .801 31. .879 48. ,740 1. .00 54. .09 6

ATOM 1946 CB GLN A 275 37, .513 31, .251 48, .481 1. ,00 27, .55 6 ATOM 1947 CA GLN A 275 36, .966 30, .920 47. .124 1. ,00 31, .54 6

ATOM 1948 C GLN A 275 36. .688 29. .412 47. .422 1. ,00 38. .56 6 ATOM 1949 0 GLN A 275 37.587 28.549 47.205 1.00 37.01

ATOM 1950 OE GLN A 275 36.105 29.125 48.479 1.00 31.65

Claims

1. 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 the parent protein,

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

2. The method according to claim 1, wherein the sequences of step a) are obtained by screening a random peptide display package library with antibodies raised against any protein of inter- est and sequencing the amino acid sequence of the antibody binding peptide, or the DNA sequence encoding the antibody binding peptide .

3. The method according to claim 2, wherein antibodies for screening the random peptide display package library are raised against the parent protein.

5 4. The method according to claims 2-3, wherein the peptide display package library is a phage display library.

5. The method according to claims 2-4, wherein the peptides of the peptide display package library are oligopeptides having io from 5 to 25 amino acids.

6. The method accoding to claim 1, wherein the antibody binding peptide sequences of step a) are obtained by screening a library of known peptides related to the primary sequence of any protein is of interest, with antibodies raised against the protein of interest .

7. The method according to any of the preceding claims, wherein epitope patterns are identified by sequence alignment of anti-

20 body binding peptide sequences and these epitope patterns are used to quide localisation of epitope sequences on the 3- dimensional structure of the parent protein.

8. The method according the any of the preceding claims, wherein 25 the epitopee area of step c) equals the epitope sequence.

9. The method according to any of the preceding claims, wherein hot spot amino acids of the parent protein are identified.

30 10. The method according to any of the preceding claims, wherein the epitope area is changed by substituting, adding and/or deleting at least one amino acid of the epitope area.

11. The method according to claim 10, wherein the epitope area is changed by substituting, adding and/or deleting at least one hot spot amino acid.

5 12. The method according to claims 10-11, wherein amino acids in the epitope area are changed by substituting and/or inserting at least one amino acid by an amino acid which render the substituted and/or inserted amino acid a target for covalent conjugation to an activated polymer.

10

13. the method according to claim 12, wherein the amino acid for substitution and/or insertion is selected from the group consisting of K, C, D, E.

is 14. The method according to claim 12, wherein the molecule for covalent conjugation is selected from the group of activated synthetic or natural polymers.

15. The method according to claim 14, wherein the activated syn- 20 thetic polymer is a polyethylene glycol .

16. The method according to any of the preceding claims, wherein the immunogenicity is measured by competitive ELISA.

5 17. The method according to any of the preseding claims, wherein the protein variant has reduced allergenicity.

18. The method according to claim 17, wherein the allergenicity of the protein variant is below 75%, preferably below 50%, more

30 preferably below 25% of the allergenicity of the parent protein.

19. The method according to any of the preceding claims, wherein the parent protein is an enzyme or an environmental allergen or a pharmaceutical protein.

20. The method according to claim 19, wherein the enzyme is selected from the group consisting of glycosyl hydrolases, carbohydrases, peroxidases, proteases, lipolytic enzymes, phytases, polysaccharide lyases, oxidoreductases, transglutaminases and glucoseisomerases .

21. The method according to claim 19, wherein the environmental allergen is selected from the group consisting of pollen, dust mites, mammals, venoms, fungi, food allergens or other plant allergens .

22. A protein variant obtainable by a method according to claims 1-21.

23. A protein variant, wherein 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 area of the parent protein.

24. The protein variant according to claim 23 having modified immunogenicity as compared to its parent protein.

25. A protein variant according to claims 22-24, wherein the epitope areas are defined on the parent protein structure by being localised less than 5 A from any of the following epitope patterns: P > S/T D P G; P > > D A G; > P > R D T G; P > S/T D P G; > R Y > K/R; > R S A; > G > > A G; V H > G >; A > I D P R/K; A R > A; Q > Y > D >; > P > > A P > S; R/K R F > N; D/E Q I F F T; A > > > > Y P >; L > G R S; R P P R; > E Y; > P > > P A P > S; > K L > >; K Q S; > K L > >; Y l > K L; R Q > > D/E; N > > E L.

26. The protein variant according to claims 22 or 23, wherein the epitope areas correspond to antibody binding peptide sequences reactive to antibodies raised against the parent protein.

27. The protein variant according to claims 22-26, wherein the epitope pattern is a IgE epitope pattern.

28. The protein variant according to claims 22-27, wherein at least one hot spot amino acid is substituted or deleted.

29. The protein variant according to claims 22-28, wherein the allergenicity of the protein variant is below 75%, preferable below 50%, more preferably below 25% of the allergenicity of the parent protein.

30. The protein variant according to claims 22-29, wherein the protein variant is an environmental allergen, preferable an allergen selected from the group consisting of pollen, dust mites, mammals, venoms, fungi, food allergens or other plant allergens.

31. The protein variant according to claims 22-29, wherein the protein variant is an antifungal peptide or antimicrobial peptide .

32. The protein variant according to claim 30, wherein the allergen is pollen allergen comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 6:

Position T 10 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, W, Y;

Position V 12 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, W, Y; Position P 14 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, , Y;

Position A 16 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y; Position R 17 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position K 20 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y;

Position L 24 to A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y;

Position F 30 to A, C, D, E, G, H, I, K, L, M, N,

P, Q, R, S, T, W, Y;

Position P 31 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, W, Y; Position K 32 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, , Y;

Position A 34 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position P 35 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y;

Position Q 36 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, W, Y;

Position A 37 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position S 39 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, , Y;

Position S 40 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, , Y;

Position E 42 to A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;

Position S 57 to A, C, D, E, F, G, H, I, K, L, M,

P, Q, R, T, V, W, Y;

Position F 58 to A, C, D, E, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position P 59 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, v, w, Y,

Position E 60 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, v, W, Y, Position G 61 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, s, T, v, W, Y,

Position L 62 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, s, T, v, , Y,

Position P 63 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, s, T, v, w, Y,

Position F 64 to A, C, D, E, G, H, I, K, L, M,

N, P, Q, R, s, T, V, w, Y;

Position K 65 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, s, T, v, , Y Position T 77 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, s, v, , Y

Position F 79 to A, C, D, E, G, H, I, K, L, M, N,

Position P 90 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, s, T, v, , Y

Position D 93 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, , Y

Position V 105 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, s, T, W, Y Position A 106 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, , Y

Position T 107 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, s, v, , Y

Position D 10S to A, C, E, F, G, H, I, K, L, M, N, P, Q, R, s, T, v, , Y

Position G 110 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, s, T, v, w, Y

Position K 123 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, s, T, v, , Y Position E 127 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position K 129 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, , Y; Position E 131 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position S 136 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, W, Y;

Position G 140 to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;

Position L 143 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, S, T, V, W, Y;

Position R 145 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y; Position S 149 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, , Y;

Position Y 150 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, W;

Position L 152 to A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, , Y;

Position A 153 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position D 156 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position Y 158 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, ;

33. The protein variant according to claim 32, wherein the pollen allergen comprises one or more of the following substitu- tions:

position P31 to A, G, L, or S; position A34 to D, E, F, H, K, N, P, Q, R, W, or Y; position P35 to A, G, L, or S ; position A37 to D, E, F, H, K, N, P, Q, R, , or Y position S39 to D, E, F, H, K, N, P, Q, R, W, or Y position S40 to D, E, F, H, K, N, P, Q, R, W, or Y position P59 to A, G, L, or S; position L62 to D, E, F, H, K, N, P, Q, R, W, or Y; position P63 to A, G, L, or S.

34. The allergen according to claims 32-33, wherein the allergen has at least 81%, preferably at least 85%, more preferably at least 90%, even more preferably at least 96%, most preferably at least 98% homology to SEQ ID NO 6 .

35. The pollen allergen according to claim 34, wherein the allergen has the amino acid sequence of SEQ ID NO 6.

36. The protein variant according to claim 30, wherein the allergen is mite allergen comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 7:

Position D 1 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position Q 2 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, W, Y; Position N 11 to A, C, D, E, F, G, H, I, K, L, M,

P, Q, R, S, T, V, W, Y;

Position E 12 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position K 14 to A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, , Y;

Position K 15 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y;

Position D 19 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y; Position G 20 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, s, T, V, w, Y

Position H 30 to A, C, D, E, F, G, I, K, L, M, N,

P, Q, R, s, T, V, , Y Position R 31 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, s, T, V, , Y

Position G 32 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, s, T, V, W, Y

Position P 34 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, s, T, V, W, Y

Position T 36 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, s, V, W, Y

Position L 37 to A, C, D, E, F, G, H, I, K, M,

N, P, Q, R, s, T, V, W Y; Position E 38 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, w, Y

Position A 39 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, W, Y,

Position L 40 to A, C, D, E, F, G, H, I, K, M, N, P, Q, R, s, T, v, , Y,

Position D 59 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, , Y,

Position L 61 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, s, T, v, w, Y Position E 62 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, w, Y

Position D 64 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, w, Y,

Position N 71 to A, C, D, E, F, G, H, I, K, L, M, P, Q, R, s, T, v, , Y₍

Position H 74 to A, C, D, E, F, G, I, K, L, M, N,

P, Q, R, s, T, v, W, Y_;

Position F 75 to A, C, D, E, G, H, I, K, L, M, N,

P, Q, R, s, T, v, , Y Position P 79 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, , Y;

Position Q 85 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, W, Y; Position D 87 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position Y 90 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, ;

Position T 91 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, , Y;

Position 92 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, Y;

Position N 93 to A, C, D, E, F, G, H, I, K, L, M,

P, Q, R, S, T, V, W, Y; Position P 95 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, W, Y;

Position K 96 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y;

Position I 97 to A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y;

Position A 98 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position P 99 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, W, Y; Position S 101 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, W, Y;

Position E 102 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position T 123 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, , Y;

Position K 126 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y;

Position R 128 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, , Y; Position D 129 to A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, , Y;

37. The mite allergen according to claims 36, wherein the aller- gen has at least 81%, preferably at least 85%, more preferably at least 90%, even more preferably at least 96%, most preferably at least 98% homology to SEQ ID NO 7.

38. The mite allergen according to claim 37, wherein the aller- gen has the amino acid sequence of SEQ ID NO 7.

39. The protein variant according to claim 30, wherein the allergen is mite allergen comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 8 :

Position L 17 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, S, T, V, , Y;

Position P 19 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, , Y;

Position G 20 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position P 26 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, W, Y; Position I 28 to A, C, D, E, F, G, H, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position H 30 to A, C, D, E, F, G, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position R 31 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y;

Position P 34 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, W, Y;

Position F 35 to A, C, D, E, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position Q 36 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, W, Y;

Position K 55 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, , Y; Position A 56 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position S 57 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, W, Y;

Position D 59 to A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, , Y;

Position G 60 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position L 61 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, S, T, V, , Y; Position E 62 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position D 64 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position P 66 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, , Y;

Position D 69 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position K 89 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, , Y; Position Y 90 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, W;

Position T 91 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, W, Y;

Position 92 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y

Position P 95 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, , Y;

Position K 96 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y; Position I 97 to A, C, D, E, F, G, H, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position P 99 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, W, Y; Position K 100 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y;

Position E 102 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position N 103 to A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y;

Position T 123 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, , Y;

Position A 125 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position R 128 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, , Y;

40. The mite allergen according to claims 39, wherein the allergen has at least 81%, preferably at least 85%, more preferably at least 90%, even more preferably at least 96%, most preferably at least 98% homology to SEQ ID NO 8.

41. The mite allergen according to claim 40, wherein the allergen has the amino acid sequence of SEQ ID NO 8.

42. The protein variant according to claim 30, wherein the allergen comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO 9 :

Position V 1 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, Y;

Position E 9 to A, C, D, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, , Y;

Position K 10 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y; Position N 13 to A, C, D, E, F, G, H, I, K, L, M,

P, Q, R, s, T, v, , Y;

Position E 14 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, w, Y; Position K 15 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, s, T, v, W, Y;

Position H 16 to A, C, D, E, F, G, I, K, L, M, N,

P, Q, R, s, T, v, , Y;

Position A 18 to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, s, T, v, W, Y;

Position R 34 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, s, T, v, W, Y;

Position H 36 to A, C, D, E, F, G, I, K, L, M, N,

P, Q, R, s, T, v, w, Y; Position G 37 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, s, T, v, , Y;

Position s 38 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, v, , Y;

Position w 41 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, s, T, V, Y;

Position V 42 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, s, T, W, Y;

Position A 43 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, , Y; Position F 54 to A, C, D, E, G, H, I, K, L, M, N,

P, Q, R, s, T, v, w,

Position D 55 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, w, Y;

Position S 56 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, v, , Y;

Position E 57 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, , Y;

Position P 59 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, s, T, v, , Y; Position L 60 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, S, T, V, , Y;

Position Q 61 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, , Y; Position P 63 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, , Y;

Position R 67 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position L 69 to A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y;

Position D 79 to A, C, E, F, G, H, I, K, L, , N,

P, Q, R, S, T, V, W, Y;

Position E 84 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position K 85 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y;

Position T 87 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, , Y;

Position P 94 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y;

43. The allergen according to claims 42, wherein the allergen has at least 81%, preferably at least 85%, more preferably at least 90%, even more preferably at least 96%, most preferably at least 98% homology to SEQ ID NO 9.

44. The allergen according to claim 43, wherein the allergen has the amino acid sequence of SEQ ID NO 9.

45. The protein variant according to claim 30, wherein the al- lergen comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO 12 :

Position I 1 to A, C, D, E, F, G, H, K, L, , N, P, Q, R, S, T, V, , Y; Position D 18 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position D 41 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position E 43 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position K 65 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, , Y;

Position Y 70 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W;

Position K 113 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y;

Position G 114 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y; Position S 116 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, W, Y;

Position P 119 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, W, Y;

Position E 120 to A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, , Y;

Position L 122 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, S, T, V, W, Y;

Position K 124 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y; Position Q 126 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, , Y;

Position Q 127 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, , Y;

Position S 130 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y;

Position R 132 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position I 139 to A, C, D, E, F, G, H, K, L, M, N,

P, Q, R, S, T, V, , Y; Position I 143 to A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y;

46. The allergen according to claims 45, wherein the allergen has at least 81%, preferably at least 85%, more preferably at least 90%, even more preferably at least 96%, most preferably at least 98% homology to SEQ ID NO 12.

47. The allergen according to claim 46, wherein the allergen has the amino acid sequence of SEQ ID NO 12.

48. The protein variant according to claim 30, wherein the allergen is a mammal allergen comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 13 :

Position S 9 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, W, Y;

Position S 12 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y;

Position Y 16 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, W;

Position D 23 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position V 40 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, W, Y;

Position R 42 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position A 43 to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;

Position L 44 to A, C, D, E, F, G, H, I, K, M,

N, P, Q, R, S, T, V, , Y;

Position Y 50 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, W; Position D 69 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position N 80 to A, C, D, E, F, G, H, I, K, L, M,

P, Q, R, S, T, V, W, Y; Position Y 84 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, ;

Position P 110 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, W, Y;

Position Q 112 to A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, Y;

Position E 120 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position P 121 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, W, Y; Position D 122 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position E 129 to A, C, D, F, G, H, I, K, L, M, N, P, Q, R, T, V, , Y;

Position K 133 to A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y;

Position G 139 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position K 142 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y; Position Q 156 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, W, Y;

Position L 157 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, S, T, V, W, Y;

Position R 158 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y;

Position G 159 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

49. The allergen according to claims 48, wherein the allergen has at least 81%, preferably at least 85%, more preferably at least 90%, even more preferably at least 96%, most preferably at least 98% homology to SEQ ID NO 13.

50. The allergen according to claim 49, wherein the allergen has the amino acid sequence of SEQ ID NO 13.

51. The protein variant according to claim 30, wherein the al- lergen comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO 15 :

Position K 1 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y; Position S 24 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, , Y;

Position E 35 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position R • 45 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y;

Position T 47 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, , Y;

Position D 52 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y; Position Y 53 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, W;

Position N 59 to A, C, D, E, F, G, H, I, K, L, M,

P, Q, R, S, T, V, W, Y;

Position R 61 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y;

Position W 62 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, Y;

Position W 63 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, Y; Position N 65 to A, C, D, E, F, G, H, I, K, L, M,

P, Q, R, S, T, V, W, Y;

Position D 66 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y; Position G 67 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position P 70 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, , Y;

Position S 72 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y;

Position R 73 to A, G, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position L 75 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, S, T, V, , Y; Position I 78 to A, C, D, E, F, G, H, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position P 79 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, W, Y;

Position S 81 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, , Y;

Position A 82 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position L 84 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, S, T, V, W, Y; Position T 118 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, W, Y;

Position R 125 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position G 126 to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, , Y;

Position R 128 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position L 129 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, S, T, V, W, Y;

52. The allergen according to claims 51, wherein the allergen has at least 81%, preferably at least 85%, more preferably at least 90%, even more preferably at least 96%, most preferably at least 98% homology to SEQ ID NO 15.

53. The allergen according to claim 52, wherein the allergen has the amino acid sequence of SEQ ID NO 15.

54. The protein variant according to claim 30, wherein the allergen comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO 16:

Position T 4 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, Y;

Position D 28 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position V 31 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, W, Y; Position Q 40 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, W, Y;

Position F 41 to A, C, D, E, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position K 42 to A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y;

Position D 44 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position E 45 to A, C, D, F, G, ,H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position A 47 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position A 48 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position K 51 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, s, T, v, w, Y

Position D 54 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, W, Y Position s 58 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, v, , Y

Position P 61 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, s, T, v, W, Y

Position T 62 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, s, v, w, Y

Position H 65 to A, C, D, E, F, G, I, K, L, M, N,

P, Q, R, s, T, v, W, Y

Position G 68 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, s, T, v, , Y Position K 70 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, s, T, v, W, Y

Position D 143 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, , Y,

Position G 146 to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, s, T, v, , Y

Position R 148 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, s, T, v, , Y

Position s 173 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, v, W, Y_; Position A 178 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, w, Y

Position K 181 to A, C, D, E, F, G, H, I, L, M,

N, P, Q, R, s, T, v, w Y;

Position D 184 to A, C, E, F, G, H, I, K, L, M, N, P, Q, R, s, T, v, , Y

Position E 185 to A, C, D, F, G, H, I, K, L, M, N,

Position P 186 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, s, T, v, w, Y Position G 187 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position S 188 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, , Y; Position A 190 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position T 192 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, , Y;

Position V 203 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, , Y;

Position I 204 to A, C, D, E, F, G, H, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position E 207 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position P 208 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, , Y;

Position G 209 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position R 213 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y;

Position K 215 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y;

Position D 236 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y; Position P 238 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, , Y;

Position T 240 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, W, Y;

Position P 241 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y;

Position G 242 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

55. The allergen according to claims 54, wherein the allergen has at least 81%, preferably at least 85%, more preferably at least 90%, even more preferably at least 96%, most preferably at least 98% homology to SEQ ID NO 16.

56. The allergen according to claim 55, wherein the allergen has the amino acid sequence of SEQ ID NO 16.

57. The protein variant according to claim 30, wherein the al- lergen comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO 17:

Position A 33 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y; Position A 36 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position T 38 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, , Y;

Position P 54 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y;

Position R 56 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position A 57 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y; Position S 58 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, W, Y;

Position V 68 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, , Y;

Position L 70 to A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y;

Position R 71 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position Y 78 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, W; Position K 80 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y;

Position K 81 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y; Position S 83 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, W, Y;

Position A 84 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position E 102 to A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, , Y;

Position K 103 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, , Y;

Position P 106 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, , Y; Position E 114 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position D 118 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position Y 119 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W;

Position I 121 to A, C, D, E, F, G, H, K, L, M, N,

P, Q, R, S, T, V, W, Y;

58. The allergen according to claims 57, wherein the allergen has at least 81%, preferably at least 85%, more preferably at least 90%, even more preferably at least 96%, most preferably at least 98% homology to SEQ ID NO 17.

59. The allergen according to claim 58, wherein the allergen has the amino acid sequence of SEQ ID NO 17.

60. The protein variant according to claim 30, wherein the allergen comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO 18 : Position w 2 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, s, T, v, Y

Position D 13 to A, C, E, F, G, H, I, K, L, M, N, s P, Q, R, s, T, V, w, Y

Position E 15 to A, C, D, F, G, H, I, K, L, M,

N, P, Q, R, s, T, V, W Y;

Position G 16 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y o Position D 28 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, s, T, V, w, Y

Position V 31 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, s, T, w, Y

Position Q 34 to A, G, D, E, F, G, H, I, K, L, M, 5 N, P, R, s, T, v, w, Y

Position Q 40 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, s, T, v, w, Y

Position L 41 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, s, T, v, w, Y 0 Position P 43 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, s, T, v, w, Y

Position Q 44 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, s, T, v, w, Y

Position D 47 to A, C, E, F, G, H, I, K, L, M, N, 5 P, Q, R, s, T, v, w, Y,

Position K 50 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, s, T, v, , Y

Position K 51 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, s, T, v, , Y 0 Position E 54 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, w, Y

Position G 57 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, s, T, v, w, Y Position A 60 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position T 62 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, , Y; Position G 67 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position G 68 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position E 69 to A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;

Position Y 71 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, W;

Position I 74 to A, C, D, E, F, G, H, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position Q 75 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, , Y;

Position Q 78 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, W, Y;

Position R 83 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y;

Position K 85 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y;

Position G 87 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y; Position P 88 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, , Y;

Position N 97 to A, C, D, E, F, G, H, I, K, L, M,

P, Q, R, S, T, V, , Y;

Position D 106 to A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, , Y;

Position P 108 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, , Y;

Position T 110 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, W, Y; Position R 120 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position D 123 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y; 5 Position Y 124 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, W;

Position E 127 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position E 129 to A, C, D, F, G, H, I, K, L, M, N, io P, Q, R, S, T, V, W, Y;

61. The allergen according to claims 60, wherein the allergen has at least 81%, preferably at least 85%, more preferably at least 90%, even more preferably at least 96%, most preferably at

15 least 98% homology to SEQ ID NO 18.

62. The allergen according to claim 61, wherein the allergen has the amino acid sequence of SEQ ID NO 18.

0 63. The protein variant according to claim 30, wherein the allergen comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO 19:

Position T 28 to A, C, D, E, F, G, H, I, K, L, M,

25 N, P, Q, R, S, V, W, Y;

Position T 31 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, , Y;

Position A 33 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; 30 Position G 34 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position A 36 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position T 53 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, W, Y;

Position A 54 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position R 56 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position G 64 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position T 65 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, Y;

Position R 66 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, , Y;

Position V 68 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, W, Y; Position R 71 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position D 74 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position Y 78 to A, C, D, E, F, G, H, I, K, L, M, position S 83 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y; Position A 84 to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, , Y; Position N 101 to A, C, D, E, F, G, H, I, K, L, M,

P, Q, R, S, T, V, W, Y;

Position E 102 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position K 103 to A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y;

Position Q 105 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, W, Y;

Position P 106 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, W, Y; Position T 108 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, , Y;

Position K 115 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y; Position Y 119 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, W;

Position T 133 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, W, Y;

Position V 136 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, Y;

Position G 137 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position D 150 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position T 153 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, , Y;

Position A 158 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position A 161 to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;

Position A 169 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position K 175 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y; Position D 176 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position R 181 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position D 199 to A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, , Y;

Position R 200 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position K 206 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y; Position G 207 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position S 208 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, W, Y; Position A 209 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position K 215 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y;

Position E 227 to A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;

Position K 228 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y;

Position I 229 to A, C, D, E, F, G, H, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position P 231 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, W, Y;

Position G 232 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position T 233 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, , Y;

Position N 236 to A, C, D, E, F, G, H, I, K, L, M,

P, Q, R, S, T, V, W, Y;

Position E 239 to A, C, D, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, , Y; Position D 243 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position Y 244 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, ;

Position I 246 to A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y;

Position G 247 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position Q 248 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, W, Y; Position G 249 to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;

64. The allergen according to claims 63, wherein the allergen has at least 81%, preferably at least 85%, more preferably at least 90%, even more preferably at least 96%, most preferably at least 98% homology to SEQ ID NO 19.

65. The allergen according to claim 64, wherein the allergen has the amino acid sequence of SEQ ID NO 19.

66. The protein variant according to claim 30, wherein the allergen comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO 20:

Position S 1 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, , Y;

Position Y 5 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, W; Position E 8 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position H 9 to A, C, D, E, F, G, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position L 12 to A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y;

Position E 47 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position E 48 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position E 70 to A, G, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position A 71 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position R 76 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, S, T, V, W, Y;

Position K 78 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y; Position G 80 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position S 81 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, W, Y;

Position K 88 to A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y;

Position G 90 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position Q 91 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, W, Y; Position E 99 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position E 100 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position P 101 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y;

Position V 102 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, W, Y;

Position T 103 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, W, Y; Position P 104 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, , Y;

Position G 105 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position Q 106 to A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, Y;

Position E 112 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position D 116 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position Y 117 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, W;

Position I 119 to A, C, D, E, F, G, H, K, L, M, N,

P, Q, R, S, T, V, , Y; Position D 120 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position Q 121 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, S, T, V, , Y;

Position G 122 to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, , Y;

Position L 123 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, S, T, V, W, Y;

67. The allergen according to claims 66, wherein the allergen has at least 81%, preferably at least 85%, more preferably at least 90%, even more preferably at least 96%, most preferably at least 98% homology to SEQ ID NO 20.

68. The allergen according to claim 67, wherein the allergen has the amino acid sequence of SEQ ID NO 20.

69. The protein variant according to claim 30, wherein the allergen comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO 21:

Position L 4 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, S, T, V, W, Y;

Position Y 6 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, W; Position Y 17 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, ;

Position S 20 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, W, Y; Position S 31 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, W, Y;

Position K 32 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, , Y;

5 Position A 33 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position K 37 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y_;

io

70. The allergen according to claims 69, wherein the allergen has at least 81%, preferably at least 85%, more preferably at least 90%, even more preferably at least 96%, most preferably at least 98% homology to SEQ ID NO 21.

15 71. The allergen according to claim 70, wherein the allergen has the amino acid sequence of SEQ ID NO 21.

72. The protein variant according to claim 30, wherein the allergen comprises one or more of the following substitutions cor- 20 responding to any of the following in SEQ ID NO 22 :

Position N 6 to A, C, D, E, F, G, H, I, K, L, M,

P, Q, R, S, T, V, W, Y;

Position C 9 to A, D, E, F, G, H, I, K, L, M, N,

25 P, Q, R, S, T, V, W, Y;

Position K 23 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y;

Position Y 24 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, W;

30 Position G 25 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position S 26 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, W, Y; Position L 27 to A, C, D, E, F, G, H, I, K, M, N,

Position K 28 to A, G, D, E, F, G, H, I, L, M, N,

P, Q, R, s, T, v, W, Y; Position P 29 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, s, T, v, ,

Position K 34 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, s, T, v, W, Y;

Position V 35 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, s, T, , Y;

Position Y 39 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, s, T, V, ;

Position G 40 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, S, T, v, w, Y; Position L 41 to A, C, D, E, F, G, H, I, K, M, N,

P, ^Q, R, s, T, v, w, Y;

Position K 43 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, s, T, v, W, Y;

Position E 45 to A, C, D, F, G, H, I, K, L, M, N, P, Q, R, s, T, v, W, Y;

Position Q 47 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, s, T, v, W, Y;

Position D 48 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, W, Y; Position L 50 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, s, T, v, w, Y;

Position K 51 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, s, T, v, , Y;

Position E 52 to A, C, D, F, G, H, I, K, L, M, N, P, Q, R, s, T, v, w, Y;

Position D 55 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, W,

Position Q 58 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, s, T, v, , Y; Position K 59 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, s, T, v, w, Y

Position R 62 to A, C, D, E, F, G, H, I, K, L,' M,

N, P, Q, s, T, v, w, Y Position G 71 to A, C, D, E, F, H, I, K, L, M, N,

P, Q, R, s, T, v, w, Y

Position P 72 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, s, T, v, w, Y

Position P 74 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, s, T, v, , Y

Position P 75 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, s, T, v, , Y

Position V 83 to A, C, D, E, F, G, H, I, K, L, M, Position N 85 to A, C, D, E, F, G, H, I, K, L, M,

P, Q, R, s, T, v, , Y

Position D 86 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, s, T, v, w, Y

Position E 87 to A, C, D, F, G, H, I, K, L, M, N, P, Q, R, s, T, v, w, Y

Position Y 90 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, s, T, V, W

Position Q 93 to A, C, D, E, F, G, H, I, K, L, M,

N, P, R, s, T, v, , Y, Position L 120 to A, C, D, E, F, G, H, I, K, M, N,

P, Q, R, s, T, v, w, Y

Position T 121 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, s, T, V, w, Y;

Position G 122 to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, s, T, v, w, Y,

Position S 123 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, v, w, Y

Position T 124 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, s, v, w, Y Position A 125 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position A 126 to C, D, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y; Position Y 128 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, W;

Position D 130 to A, C, E, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position D 140 to A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;

Position P 147 to A, C, D, E, F, G, H, I, K, L, M,

N, Q, R, S, T, V, W, Y;

Position K 148 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y; Position K 150 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, , Y;

Position S 152 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, T, V, , Y;

Position G 153 to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;

Position F 156 to A, C, D, E, G, H, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position K 158 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y; Position H 161 to A, C, D, E, F, G, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position I 181 to A, C, D, E, F, G, H, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position E 183 to A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;

Position K 184 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, W, Y;

Position W 185 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, T, V, Y; Position H 186 to A, C, D, E, F, G, I, K, L, M, N,

P, Q, R, S, T, V, W, Y;

Position N 199 to A, C, D, E, F, G, H, I, K, L, M,

P, Q, R, S, T, V, W, Y; Position K 201 to A, C, D, E, F, G, H, I, L, M, N,

P, Q, R, S, T, V, , Y;

Position N 202 to A, C, D, E, F, G, H, I, K, L, M,

P, Q, R, S, T, V, , Y;

Position E 203 to A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;

Position E 204 to A, C, D, F, G, H, I, K, L, M, N,

P, Q, R, S, T, V, , Y;

Position T 208 to A, C, D, E, F, G, H, I, K, L, M,

N, P, Q, R, S, V, W, Y;

73. The allergen according to claims 72, wherein the allergen has at least 81%, preferably at least 85%, more preferably at least 90%, even more preferably at least 96%, most preferably at least 98% homology to SEQ ID NO 22.

74. The allergen according to claim 73, wherein the allergen has the amino acid sequence of SEQ ID NO 22.

75. The protein variant according to claims 22-29, wherein the protein variant is an enzyme.

76. The protein variant according to claim 75, wherein the enzyme is a protease, a lipolytic enzyme, a carbohydrase or a oxidoreductase .

77. The protein variant according to claim 76, wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 10: Position -6 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, , Y, insertion, deletion;

Position -5 to A, C, D, E, F, G, H, I, K, L, M, N, P,

5 Q, R, S, T, V, W, Y, insertion, deletion;

Position -4 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, W, Y, insertion, deletion;

Position -2 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, , Y, insertion, deletion; io Position 3a to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, W, Y, insertion, deletion;

Position 28a to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, W, Y, insertion, deletion;

Position 44a to A, C, D, E, F, G, H, I, K, L, M, N, P, is Q, R, S, T, V, W, Y, insertion, deletion;

Position 44b to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, W, Y, insertion, deletion;

Position 139 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, , Y, insertion, deletion;

20 Position 148 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, W, Y, insertion, deletion;

Position 149 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, W, Y, insertion, deletion;

Position 264a to A, C, D, E, F, G, H, I, K, L, M, N, P,

25 Q, R, S, T, V, , Y, insertion, deletion;

78. The protein variant according to claim 76, wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 30 10 :

Position -1 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, position 1 to V, L, I, W, M, F, Y, S, T, R; Position 2 to G, V, I, M, F, N, Q, Y, S, T, H;

Position 3 to W, M, F, N, Q, Y, S, D, E, R, H;

Position 4 to V, L, W, M, F, Y, R;

Position 5 to V, L, I, W, M, F, N, Q, Y, T, R, H;

Position 6 to G, V, L, I, , P, M, N, Q, T, D, E, R,

position 9 to G, V, L, I, , P, M, F, Q, Y, S, T, R,

position 10 to G, A, V, I, W, P, M, N, Q, Y, S, T, D,

E, R;

Position 12 to G, A, V, L, I, , M, F, N, Q, Y, S, T,

D, E;

Position 14 to V, L, I, W, P, M, F, N, Q, Y, T, R, H;

Position 15 to G, A, V, L, I, , P, M, F, N, Q, Y, S,

T, E, H;

Position 17 to G, A, V, I, W, P, M, F, Y, H;

Position 18 to G, A, L, I, , P, M, F, N, Q, Y, T, D,

position 19 to A, V, I, W, M, F, N, Y, S, T, D, R, H;

Position 20 to G, V, L, I, , M, F, N, Q, Y, S, T, D, E;

Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H;

Position 22 to G, V, L, I, , M, F, Y, S, T;

Position 24 to G, V, L, I, W, M, F, N, Q, Y, S, D, E,

R;

Position 25 to G, A, V, L, I, , M, F, N, Q, Y, S, T,

D, E, R, H;

Position 27 to G, L, I, , P, M, F, Y, T, H;

Position 38 to v, L, I, , M, F, N, Q, Y, T, H;

Position 39 to G, A, V, L, I, W, M, F, N, Q, Y, T, D, E, R, H;

Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H;

Position 42 to G, A, L, , C, M, F, N, Q, Y, S, T, D,

E, R, H; Position 43 to G, L, H;

Position 44 to G, V, L, I, W, P, M, F, Y, S, T;

Position 45 to G, V, L, I, W, P, M, F, N, Q, Y, S, T,

D, E, R, H;

Position 46 to G, A, L, I, , P, M, F, Y, H;

Position 47 to G, A, V, L, I, , P, M, F, N, Q, Y, S,

T, D, E, R, H;

Position 48 to A, L, I, P, M, F, N, Y, D, H;

Position 49 to G, A, V, I, W, P, M, F, N, Q, Y, S, T,

D, E, R, H;

Position 50 to G, A, W, M, N, Q, Y, S, T, D, E, H;

Position 51 to V, L, I, W, M, F, N, Y, R;

Position 52 to V, L, I, W, M, F, Y, S, T, R;

Position 53 to A, V, L, I, W, M, F, N, Q, Y, S, D, E,

position 54 to V, L, I, W, M, F, S, R;

Position 55 to G, A, V, L, I, , C, M, F, N, Q, Y, T,

D, E, R, K, H;

Position 56 to G, V, L, I, W, M, F, N, Q, Y, S, T, H;

Position 57 to G, A, V, L, I, W, M, F, N, Q, Y, S, T,

D, E, R, K, H;

Position 58 to L, W, M, F, N, Y, R;

Position 59 to A, V, L, I, C, T, H;

Position 61 to V, L, I, W, M, F, Y;

Position 62 to G, A, L, W, M, F, N, Y, R;

Position 64 to G, V, L, I, W, P, C, M, F, N, Q, Y, S,

Position 75 to L;

Position 79 to I;

Position 80 to G;

Position 87 to A, V, L, I, W, M, F, Q, Y, S, T, D, E,

position ^■89 to G G,, V, L, I, , P, F, N, Y, T, E; Position 91 to G, A, V, L, I, , P, M, N, Y, S, T, D,

E , R, H;

Position 98 to A

Position 99 to V L, I, , M, F, Q, Y, H; Position 100 to G V, L, I, W, M, F, Y, R, H;

Position 101 to V I, W, M, F, N, Q, Y, H;

Position 102 to V L, I, W, M, F, Y, R, H, G;

Position 108 to I

Position 109 to N Position 112 to E

Position 113 to W

Position 115 to I

Position 117 to N

Position 118 to N Position 126 to L

Position 127 to G A, V, I, , M, F, Y, R, H, L;

Position 128 to I ;

Position 129 to w

Position 130 to w F , Y , R ; Position 131 to w Y, R;

Position 132 to L W, M, F, Y, S, H;

Position 133 to A L, I, W, M, F, Y, R;

Position 134 to L I, W, F, N, Q, Y, R, H;

Position 136 to G A, , P, N, Y, S, T, D, E, H; Position 137 to G A, V, I, W, P, M, N, Y, H;

Position 140 to G A, V, L, I, W, P, M, F, N, Q, Y, S,

position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H;

Position 143 to V, L, I, P, M, F, N, Y, R; Position 144 to L, , P, M, F, N, Q, Y, S, D, E, R, H;

Position 145 to G, V, L, I, , M, F, Q, Y, D, E, R, H;

Position 146 to G, A, , L, I, , M, F, N, Q, Y, T, D,

E , R, H;

Position 155 to V, L, I, W, M, F, Y, R; Position 156 to V, I, W, F, R;

Position 157 to G, A, V, L, I, W, M, F, Y, T, R, H;

Position 158 to V, L, I, W, M, F, Y;

Position 159 to A, , M, Y, T, R, H;

Position 160 to W, M, F, Y, R, H;

Position 161 to I, W, M, F, Y, H;

Position 167 to R, K;

Position 171 to D;

Position 172 to G, A, V, L, I, S, T, H;

Position 173 to G, A, V, L, I, W, M, F, N, Q, Y, S, T,

position 181 to G, A, V, L, I, , C, M, F, Q, Y, T, D,

R, K, H;

Position 182 to A, V, L, I, , C, M, F, N, Q, Y, S, T,

position 183 to G, A, V, L, W, C, M, F, N, Q, Y, S, T,

E , R, H;

Position 184 to A, V, L, I, , C, M, F, N, Q, Y, T, E,

position 185 to G, A, V, L, I, , C, M, F, N, Q, Y, T,

position 186 to G, A, V, L, , M, F, N, Q, Y, S, T, D,

E , R, H;

Position 188 to G, A, V, L, W, F, S, R, K;

Position 189 to W, F;

Position 191 to A, V, L, I, W, M, F, Y, T, R, H;

Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H;

Position 194 to W, N, Q, Y, D, H;

Position 195 to W, P, Y;

Position 196 to G, A, V, L, I, , P, M, F, N, Q, Y, S,

T, D, E, R, H;

Position 203 to V, F, Y, R, H;

Position 204 to I, , M, Y, H;

Position 206 to F; Position 209 to Y, R;

Position 210 to w, F, Y;

Position 211 to L, W, M, F, Y, H;

Position 212 to v, L, I, , M, F, Y, T, R, H; Position 214 to W, Y, R;

Position 215 to A, L, I, W, M, F, Y;

Position 216 to A, L, I, , M, F, Y, R;

Position 217 to W, R;

Position 218 to G, A, L, W, P, M, F, Y, R, H; Position 221 to S;

Position 236 to S;

Position 240 to N;

Position 241 to W;

Position 243 to N; Position 245 to Q;

Position 247 to G, V, I, W, P, F, Y, S, T, R;

Position 248 to , P, F, Y, E, R, H;

Position 249 to L, , P, F, S, D, E, H;

Position 251 to G, L, I, W, P, M, F, Y, H; Position 252 to G, A, , P, N, Q, Y, T, E, R, H;

Position 254 to G, ¥, L, I, , M, F, N, Q, Y, S, D, E,

position 255 to G, L, , M, F, N, Y, T, D, H;

Position 256 to G, A, V, L, I, W, M, F, Q, Y, S, T, D,

position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, position 258 to G, A, V, L, I, W, C, M, F, N, Q, Y, S,

T, E, K, H; Position 259 to A, V, I, W, M, F, N, Q, Y, S, T, E, R;

Position 260 to L, I, , M, F, Y, T, H;

Position 261 to L, N, S, H;

Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D,

E, R, H; Position 263 to G, A, V, L, I, P, C, M, N, Q, Y, S, T, R, K; Position 265 to V, L, I, , M, F, Y; Position 269 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, position 271 to A, L, I, , P, M, F, N, Y, S, T, R, H; Position 272 to G, A, V, L, I, W, P, M, F, N, Q, Y, T, D, E, H; Position 275 to G, A, V, L, I, W, M, F, N, Y, T, D; 10

79. The protein variant according to claim 76, wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 15 10:

Position -1 to Deletion;

Position 9 to Insertion deletion

Position 10 to Insertion deletion

20 Position 12 to Insertion deletion

Position 14 to Insertion deletion

Position 15 to Insertion deletion

Position 17 to Insertion deletion

Position 18 to Insertion deletion

25 Position 19 to Insertion deletion

Position 20 to Insertion deletion

Position 21 to Insertion , deletion

Position 22 to Insertion , deletion

Position 24 to Insertion , deletion

30 Position 25 to Insertion , deletion

Position 46 to Insertion , deletion

Position 47 to Insertion , deletion

Position 48 to Insertion , deletion

Position 49 to Insertion , deletion Position 50 to Insertion, deletion;

Position 51 to Insertion, deletion;

Position 52 to Insertion, deletion;

Position 53 to Insertion, deletion; Position 54 to Insertion, deletion;

Position 55 to Insertion, deletion;

Position 58 to Insertion, deletion;

Position 59 to Insertion, deletion;

Position 61 to Insertion, deletion; Position 64 to Insertion, deletion;

Position 78 to Insertion:

Position 80 to Insertion;

Position 91 to Insertion, deletion;

Position 98 to Deletion; Position 99 to Deletion;

Position 102 to Deletion;

Position 105 to Insertion;

Position 108 to Insertion;

Position 109 to Insertion; Position 112 to Insertion;

Position 113 to Insertion;

Position 115 to Insertion;

Position 116 to Insertion;

Position 117 to Insertion; Position 118 to Insertion;

Position 131 to Deletion;

Position 134 to Insertion, deletion;

Position 136 to Insertion, deletion;

Position 137 to Insertion, deletion; Position 140 to Insertion, deletion;

Position 141 to Insertion, deletion;

Position 143 to Insertion, deletion;

Position 144 to Insertion, deletion;

Position 145 to Insertion, deletion; Position 146 to Insertion, deletion;

Position 171 to Deletion

Position 172 to Deletion

Position 173 to Deletion Position 181 to Deletion

Position 182 to Deletion

Position 183 to Deletion

Position 184 to Deletion,

Position 185 to Deletion Position 186 to Deletion

Position 188 to Deletion

Position 189 to Deletion

Position 191 to Deletion

Position 192 to Deletion Position 195 to Deletion;

Position 196 to Insertion, deletion;

Position 221 to Insertion;

Position 236 to Insertion;

Position 237 to Insertion; Position 238 to Insertion;

Position 239 to Insertion;

Position 240 to Insertion;

Position 241 to Insertion;

Position 242 to Insertion; Position 243 to Insertion;

Position 244 to Insertion;

Position 245 to Insertion;

Position 247 to Insertion, deletion;

Position 248 to Insertion, deletion; Position 249 to Insertion, deletion;

Position 251 to Insertion, deletion;

Position 252 to Insertion, deletion;

Position 254 to Insertion, deletion;

Position 255 to Insertion 1, deletion; Position 256 to Insertion, deletion

Position 257 to Insertion, deletion

Position 258 to Insertion, deletion

Position 259 to Insertion, deletion Position 260 to Insertion, deletion

Position 261 to Insertion, deletion

Position 262 to Insertion, deletion

Position 263 to Insertion, deletion

Position 265 to Insertion, deletion Position 269 to Insertion, deletion

Position 271 to Insertion, deletion

Position 272 to Insertion, deletion

Position 275 to Insertion, deletion

80. The protein variant according to claim 76, wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 10:

Position 7 to G, A, V, L, I, , P, M, F, N, Q, Y, S, T, D, E, R, H; Position 8 to G, A, L, , P, C, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 13 to G, L, I, , P, M, F, N, Q, Y, S, D, E,

position 16 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, position 23 to G, A, V, L, I, , M, F, Y, E, R, H; Position 26 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, position 28 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 29 to GG,, A, V, L, I, , P, M, F, N, Q, Y, S,

T, D, E, R, K, H;

Position 33 to V, L, I, , C, M, F, N, Q, Y, R, H;

Position 35 to G, A, V, L, I, , M, F, N, Q, Y, S, T,

D, E, R, K, H;

Position 36 to V, L, I, W, P, M, F, N, Y, S, T, R, H;

Position 37 to L, I, W, M, F, N, Q, Y, S, R, H;

Position 41 to G, V, L, I, W, M, F, N, Q, Y, S, T, R,

position 60 to G, A, V, L, I, , C, M, F, Q, Y, T, D,

position 63 to G, A, V, L, I, W, M, F, Y, T, R, H;

Position 73 to A;

Position 74 to A;

Position 81 to V;

Position 82 to L;

Position 86 to G, A, V, L, I, , M, F, N, Q, Y, T, D,

position 88 to A, V, L, I, , M, F, N, Q, Y, S, T, D,

position 92 to G, A, V, L, I, W, P, M, F, N, Q, Y, S,

T, D, E, R, K, H;

Position 93 to G, A, V, L, I, W, P, M, F, N, Q, Y, S,

T, D, E, R, K, H;

Position 94 to G, V, L, I, W, P, M, F, N, Y, T, D, E,

position 96 to L, W, F, Y, R, K;

Position 97 to V, L, W, C, M, F, Y, H;

Position 111 to I;

Position 114 to A;

Position 119 to M;

Position 124 to M;

Position 135 to G, L, P, C, N, Q, T, R, H; Position 138 to GG,, A, V, L, I, , P, M, F, N, Q, Y, S,

T, D, E, R, H;

Position 142 to G, A, L, I, , P, C, M, F, N, Q, Y, S,

T, D, E, R, I _r H; Position 147 to G, A, V, L, W, M, F, N, Q, Y, S, T, D,

E, R, K, H;

Position 151 to G, V, L, I, W, P, C, M, F, N, Q, Y, S,

T, D, E, R, K, H;

Position 162 to I, W, F, Y, R; Position 163 to V, W, M, F, H;

Position 168 to G, V, L, I, W, C, M, F, N, Q, Y, S, T,

D, E, R, K, H;

Position 169 to c, E, F, G, H, I, K, L, M, N, Q, R, T,

V, W, Y; Position 174 to G, A, L, I, W, P, C, M, F, N, Q, Y, S,

T, D, E, R, K, H;

Position 176 to G, A, V, L, I, W, P, C, M, F, N, Q, Y,

S, T, D, E, R, K, H;

Position 179 to G, A, V, L, I, , P, M, F, N, Q, Y, S, T, D, E, R, K, H;

Position 187 to A, V, L, I, W, M, F, Y, R;

Position 190 to G, A, V, L, I, , C, M, F, N, Q, Y, S,

T, R, K, H;

Position 193 to G, V, L, I, W, M, F, N, Q, Y, S, T, D, E , R, H;

Position 197 to G, V, L, I, W, P, M, F, Q, Y, S, T, H;

Position 198 to G, A, L, I, W, P, C, M, F, N, Q, Y, S,

T, D, E, R, K, H;

Position 205 to W, F, Y, R, K; Position 208 to A, V, L, I, W, C, M, F, Y, T, R, K, H;

Position 219 to G, A, V, L, I, W, F, Y, R, H;

Position 222 to M;

Position 232 to A;

Position 233 to L; Position 234 to I; Position 250 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, position 267 to G, A, V, L, I, , M, F, N, Q, Y, S, T, position 268 to G, V, L, I, , C, M, N, Q, Y, S, T, D, position 270 to G, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H; Position 273 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, position 274 to W, P, M, F, N, Q, Y, T, D, E, R, H;

81. The protein variant according to claim 76, wherein the pro- tease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 10:

Position 13 to Insertion, deletion Position 16 to Insertion, deletion

Position 23 to Insertion, deletion

Position 26 to Insertion, deletion

Position 28 to Insertion, deletion

Position 29 to Insertion, deletion Position 35 to Deletion;

Position 60 to Insertion, deletion;

Position 63 to Insertion;

Position 81 to Insertion;

Position 82 to Insertion; Position 92 to Insertion, deletion

Position 93 to Insertion, deletion

Position 94 to Insertion, deletion

Position 96 to Deletion,

Position 106 to Insertion, Position 111 to Insertion,

Position 114 to Insertion,

Position 119 to Insertion,

Position 124 to Insertion, Position 138 to Insertion, deletion;

Position 142 to Insertion, deletion;

Position 147 to Insertion, deletion;

Position 151 to Insertion, deletion;

Position 174 to Insertion, deletion; Position 176 to Insertion, deletion;

Position 179 to Insertion, deletion;

Position 187 to Deletion;

Position 190 to Deletion;

Position 193 to Deletion; Position 197 to Insertion, deletion;

Position 198 to Insertion, deletion;

Position 232 to Insertion,

Position 233 to Insertion,

Position 234 to Insertion, Position 246 to Insertion,

Position 250 to Insertion, deletion;

Position 267 to Insertion, deletion;

Position 268 to Insertion, deletion;

Position 270 to Insertion, deletion; Position 273 to Insertion, deletion;

82. The protein variant according to claims 76-81, wherein the protease is a savinase-like subtilisin comprising one or more of the following substitutions corresponding to any of the fol^¬ lowing in SEQ ID NO: 10:

Position 2 to G, V, I, M, F, N, Q, Y, S, T, H, Position 3 to W, M, F, N, Q, Y, S, D, E, R, H, Position 4 to V, L, , M, F, Y, R,

Position 6 to G, V, L, I, W, P, M, N, Q, T, D, E, R, H,

Position 9 to G, V, L, I, , P, M, F, Q, Y, S, T, R, H, insertion, deletion,

Position _. 10 to G, A, V, I, W, P, M, N, Q, Y, S, T, D,

E, R, insertion, deletion,

Position 12 to G, A, V, L, I, , M, F, N, Q, Y, S, T,

D, E, insertion, deletion, Position 14 to V, L, I, , P, M, F, N, Q, Y, T, R, H, insertion, deletion,

Position 15 to G, A, V, L, I, W, P, M, F, N, Q, Y, S,

T, E, H, insertion, deletion,

Position 17 to G, A, V, I, W, P, M, F, Y, H, insertion, deletion,

Position 18 to G, A, L, I, , P, M, F, N, Q, Y, T, D,

E, H, insertion, deletion,

Position 19 to A, V, I, W, M, F, N, Y, S, T, D, R, H, insertion, deletion, Position 20 to G, V, L, I, W, M, F, N, Q, Y, S, T, D,

E, insertion, deletion,

Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion,

Position 22 to G, V, L, I, W, M, F, Y, S, T, insertion, deletion,

Position 24 to G, V, L, I, W, M, F, N, Q, Y, S, D, E,

R, insertion, deletion,

Position 25 to G, A, V, L, I, , M, F, N, Q, Y, S, T,

D, E, R, H, insertion, deletion, Position 27 to G, L, I, W, P, M, F, Y, T, H,

Position 37 to L, I, W, M, F, N, Q, Y, S, R, H,

Position 40 to V, L, I, , M, F, N, Q, Y, T, R, H,

Position 42 to G, A, L, , C, M, F, N, Q, Y, S, T, D,

E, R, H, Position 43 to G, L, H,

Position 44 to G, V, L, I, W, P, M, F, Y, S, T,

Position 45 to G, V, L, I, W, P, M, F, N, Q, Y, S, T, D_/ E _f R., H_/ Position 46 to G, A, L, I, W, P, M, F, Y, H, insertion, deletion,

Position 47 to G, A, V, L, I, W, P, M, F, N, Q, Y, S,

T, D, E, R, H, insertion, deletion,

Position 48 to A, L, I, P, M, F, N, Y, D, H, insertion, deletion,

Position 50 to G, A, , M, N, Q, Y, S, T, D, E, H, insertion, deletion,

Position 51 to V, L, I, W, M, F, N, Y, R, deletion, insertion, Position 54 to V, L, I, W, M, F, S, R, deletion, insertion,

Position 55 to G, A, V, L, I, W, C, M, F, N, Q, Y, T,

D, E, R, K, H, deletion, insertion,

Position 57 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H,

Position 58 to L, W, M, F, N, Y, R, insertion, deletion,

Position 59 to A, V, L, I, C, T, H, insertion, deletion, Position 61 to V, L, I, W, , F, Y, insertion, deletion,

Position 64 to G, V, L, I, , P, C, M, F, N, Q, Y, S,

T, D, E, R, K, H, insertion, deletion,

Position 75 to L, Position 78 to insertion,

Position 79 to I,

Position 87 to A, V, L, I, W, M, F, Q, Y, S, T, D, E, H,

Position 89 to G, V, L, I, , P, F, N, Y, T, E, Position 91 to G, A, V, L, I, W, P, M, N, Y, S, T, D,

E, R, H, insertion, deletion,

Position 98 to A, deletion,

Position 100 to G, V, L, I, W, M, F, Y, R, H, Position 101 to V, I, W, M, F, N, Q, Y, H,

Position 102 to V, L, I, , M, F, Y, R, H, G, deletion,

Position 109 to N, insertion,

Position 112 to E, insertion,

Position 113 to W, insertion, Position 116 to insertion,

Position 117 to N, insertion,

Position 126 to L,

Position 127 to G, A, V, I, W, M, F, Y, R, H, L,

Position 128 to I, W, Position 129 to W,

Position 130 to , F, Y, R,

Position 131 to W, Y, R, deletion,

Position 132 to L, , M, F, Y, S, H,

Position 133 to A, L, I, W, M, F, Y, R, Position 134 to L, I, W, F, N, Q, Y, R, H, insertion, deletion,

Position 136 to G, A, W, P, N, Y, S, T, D, E, H, insertion, deletion,

Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion,

Position 140 to G, A, V, L, I, W, P, M, F, N, Q, Y, S,

T, H, insertion, deletion,

Position 141 to G, V, L, I, , P, M, F, Q, S, D, E, H, insertion, deletion, Position 143 to V, L, I, P, M, F, N, Y, R, insertion, deletion,

Position 144 to L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion, Position 145 to G, V, L, I, W, M, F, Q, Y, D, E, R, H, insertion, deletion,

Position 146 to G, A, W, L, I, W, M, F, N, Q, Y, T, D,

E, R, H, insertion, deletion, Position 155 to V, L, I, , M, F, Y, R,

Position 156 to V, I, W, F, R,

Position 157 to G, A, V, L, I, W, M, F, Y, T, R, H,

Position 158 to V, L, I, W, M, F, Y,

Position 160 to W, M, F, Y, R, H, Position 161 to I, W, M, F, Y, H,

Position 167 to R, K,

Position 170 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, W, Y;

Position 171 to D, deletion, Position 172 to G, A, V, L, I, S, T, H, deletion,

Position 173 to G, A, V, L, I, , M, F, N, Q, Y, S, T,

E, H, deletion,

Position 181 to G, A, V, L, I, W, C, M, F, Q, Y, T, D,

R, K, H, deletion, Position 183 to G, A, V, L, W, C, M, F, N, Q, Y, S, T,

E, R, H, deletion,

Position 184 to A, V, L, I, W, C, M, F, N, Q, Y, T, E,

H, deletion,

Position 185 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion,

Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D,

E, R, H, deletion,

Position 188 to G, A, V, L, W, F, S, R, K, deletion,

Position 189 to W, F, deletion, Position 191 to A, V, L, I, W, M, F, Y, T, R, H, deletion,

Position 192 to G, L, I, , M, N, Q, Y, S, T, D, R, H, deletion,

Position 194 to W, N, Q, Y, D, H, Position 195 to W, P, Y, deletion,

Position 197 to G V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion,

Position 203 to V F, Y, R, H,

Position 206 to F

Position 209 to Y R,

Position 210 to W F, Y,

Position 212 to V L, I, W, M, F, Y, T, R, H,

Position 214 to W Y, R,

Position 216 to A L, I, W, M, F, Y, R,

Position 217 to W R,

Position 218 to G A, L, W, P, M, F, Y, R, H,

Position 221 to S insertion,

Position 236 to S insertion,

Position 237 to insertion,

Position 239 to insertion,

Position 240 to N, insertion,

Position 241 to W, insertion,

Position 242 to insertion,

Position 244 to insertion,

Position 245 to Q, insertion,

Position 247 to G, V, I, W, P, F, Y, S, T, R, insertion, deletion,

Position 248 to W, P, F, Y, E, R, H, insertion, dele- tion,

Position 251 to G, L, I, W, P, M, F, Y, H, insertion, deletion,

Position 252 to G, A, W, P, N, Q, Y, T, E, R, H, inser- tion, deletion,

Position 255 to G, L, W, M, F, N, Y, T, D, H, insertion, deletion,

Position 256 to G, A, V, L, I, W, M, F, Q, Y, S, T, D,

H, insertion, deletion, Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T,

D, E, K, H, insertion, deletion,

Position 258 to G, A, V, L, I, , C, M, F, N, Q, Y, S,

T, E, K, H, insertion, deletion, Position 259 to A, V, I, , M, F, N, Q, Y, S, T, E, R, insertion, deletion,

Position 260 to L, I, W, M, F, Y, T, H, insertion, deletion,

Position 261 to L, N, S, H, insertion, deletion, Position 262 to G, A, V, L, I, , P, F, N, Q, Y, T, D,

E, R, H, insertion, deletion,

Position 263 to G, A, V, L, I, P, C, M, N, Q, Y, S, T, R, K, insertion, deletion,

Position 265 to V, L, I, W, M, F, Y, insertion, dele- tion,

Position 271 to A, L, I, , P, M, F, N, Y, S, T, R, H, insertion, deletion,

Position 272 to G, A, V, L, I, , P, M, F, N, Q, Y, T,

D, E, H, insertion, deletion, Position 275 to G, A, V, L, I, , M, F, N, Y, T, D, insertion, deletion,

83. The protein variant according to claim 82, wherein the savinase-like subtilisin comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:

Position 6 to G, V, L, I, W, P, M, N, Q, T, D, E, R, H, Position 9 to G, V, L, I, W, P, M, F, Q, Y, S, T, R, H, insertion, deletion, Position 10 to G, A, V, I, W, P, M, N, Q, Y, S, T, D,

E, R, insertion, deletion, Position 14 to V, L, I, , P, M, F, N, Q, Y, T, R, H, insertion, deletion,

Position 15 to G, A, V, L, I, W, P, M, F, N, Q, Y, S,

T, E, H, insertion, deletion, Position 17 to G, A, V, I, , P, M, F, Y, H, insertion, deletion,

Position 18 to G, A, L, I, , P, M, F, N, Q, Y, T, D,

E, H, insertion, deletion,

Position 19 to A, V, I, W, M, F, N, Y, S, T, D, R, H, insertion, deletion,

Position 20 to G, V, L, I, W, M, F, N, Q, Y, S, T, D,

E, insertion, deletion,

Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion, Position 37 to L, I, W, M, F, N, Q, Y, S, R, H,

Position 43 to G, L, H,

Position 45 to G, V, L, I, W, P, M, F, N, Q, Y, S, T,

D, E, R, H,

Position 47 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,

Position 50 to G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion,

Position 59 to A, V, L, I, C, T, H, insertion, deletion,

Position 89 to G, V, L, I, W, P, F, N, Y, T, E, Position 91 to G, A, V, L, I, W, P, M, N, Y, S, T, D,

E, R, H, insertion, deletion,

Position 101 to V, I, , M, F, N, Q, Y, H,

Position 109 to N, insertion,

Position 112 to E, insertion, Position 113 to W, insertion,

Position 127 to G, A, V, I, , M, F, Y, R, H, L,

Position 128 to I, , Position 129 to , Position 130 to , F, Y, R,

Position 131 to W, Y, R, deletion,

Position 133 to A, L, I, , M, F, Y, R,

Position 136 to G, A, , P, N, Y, S, T, D, E, H, insertion, deletion, Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion,

Position 140 to G, A, V, L, I, , P, M, F, N, Q, Y, S, T, H, insertion, deletion,

Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion,

Position 143 to V, L, I, P, M, F, N, Y, R, insertion, deletion,

Position 144 to L, , P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion, Position 145 to G, V, L, I, W, M, F, Q, Y, D, E, R, H, insertion, deletion,

Position 146 to G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H, insertion, deletion,

Position 155 to V, L, I, W, M, F, Y, R,

Position 157 to G, A, V, L, I, W, M, F, Y, T, R, H,

Position 158 to V, L, I, , M, F, Y,

Position 160 to W, M, F, Y, R, H,

Position 161 to I, , M, F, Y, H,

Position 167 to R, K,

Position 170 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;

Position 171 to D, deletion,

Position 172 to G, A, V, L, I, S, T, H, deletion, Position 173 to G, A, V, L, I, , M, F, N, Q, Y, S, T, E, H, deletion, Position 181 to G, A, V, L, I, , C, M, F, Q, Y, T, D, R, K, H, deletion, Position 184 to A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion, Position 185 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion, Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion, Position 188 to G, A, V, L, W, F, S, R, K, deletion, Position 189 to , F, deletion, Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion, Position 194 to , N, Q, Y, D, H, Position 195 to W, P, Y, deletion, Position 197 to G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion, Position 203 to V, F, Y, R, H,

Position 210 to W, F, Y,

Position 218 to G, A, L, W, P, M, F, Y, R, H,

Position 236 to S, insertion,

Position 237 to insertion,

Position 239 to insertion,

Position 240 to N, insertion,

Position 241 to , insertion,

Position 242 to insertion,

Position 244 to insertion,

Position 245 to Q, insertion,

Position 247 to G, V, I, W, P, F, Y, S, T, R, insertion, deletion,

Position 251 to G, L, I, , P, M, F, Y, H, insertion, deletion, Position 255 to G, L, W, M, F, N, Y, T, D, H, insertion, deletion,

Position 256 to G, A, V, L, I, , M, F, Q, Y, S, T, D, H, insertion, deletion, Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T,

D, E, K, H, insertion, deletion,

Position 258 to G, A, V, L, I, , C, M, F, N, Q, Y, S, T, E, K, H, insertion, deletion,

Position 260 to L, I, W, M, F, Y, T, H, insertion, dele- tion,

Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D,

E, R, H, insertion, deletion,

Position 265 to V, L, I, W, M, F, Y, insertion, deletion, Position 271 to A, L, I, W, P, M, F, N, Y, S, T, R, H, insertion, deletion,

Position 272 to G, A, V, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion, deletion, Position 275 to G, A, V, L, I, W, M, F, N, Y, T, D, in- sertion, deletion,

84. The savinase-like subtilisin according to claims 82-83, wherein the subtilisin has at least 81%, preferably at least 96%, more preferably at least 98%, most preferably at least 99% homology to SEQ ID NO 24.

85. The savinase-like subtilisin according to claim 84, wherein the subtilisin has any of the amino acid sequence of SEQ ID NO 24, 26, 27, 28, 29, 30, 31, 32, 34, 35.

86. The protein variant according to claims 76-81, wherein the protease is a savinase-like subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10: Position 8 to G, A, L, , P, C, M, F, N, Q, Y, S, T,

D, E, R, K, H,

Position 16 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,

Position 23 to G, A, V, L, I, W, M, F, Y, E, R, H, insertion, deletion,

Position 26 to G, A, V, L, I, W, M, F, N, Q, Y, S, T,

D, E, R, H, insertion, deletion, Position 35 to G, A, V, L, I, W, M, F, N, Q, Y, S, T,

D, E, R, K, H, deletion,

Position 38 to V, L, I, W, M, F, N, Q, Y, T, H,

Position 39 to G, A, V, L, I, W, M, F, N, Q, Y, T, D,

E, R, H, Position 41 to G, V, L, I, W, M, F, N, Q, Y, S, T, R,

H,

Position 60 to G, A, V, L, I, W, C, M, F, Q, Y, T, D,

R, K, H, insertion, deletion,

Position 73 to A, Position 74 to A,

Position 80 to G, insertion,

Position 81 to V, insertion,

Position 86 to G, A, V, L, I, W, M, F, N, Q, Y, T, D,

Position 88 to A, V, L, I, W, M, F, N, Q, Y, S, T, D,

E, R, H,

Position 90 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, , Y, insertion, deletion,

Position 93 to G, A, V, L, I, , P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,

Position 108 to I, insertion,

Position 111 to I, insertion,

Position 124 to M, insertion,

Position 135 to G, L, P, C, N, Q, T, R, H, Position 142 to G, A, L, I, W, P, C, M, F, N, Q, Y, S,

T, D, E, R, K, H, insertion, deletion,

Position 147 to G, A, V, L, W, M, F, N, Q, Y, S, T, D,

E, R, K, H, insertion, deletion, Position 148 to G, A, V, L, I, W, P, C, M, F, N, Q, Y,

S, T, D, E, R, K, H, insertion, deletion,

Position 149 to G, A, V, L, I, W, P, C, M, F, N, Q, Y,

S, T, D, E, R, K, H, insertion, deletion,

Position 151 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,

Position 163 to V, W, M, F, H,

Position 168 to G, V, L, I, , C, M, F, N, Q, Y, S, T,

D, E, R, K, H,

Position 169 to C, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y,

Position 174 to G, A, L, I, W, P, C, M, F, N, Q, Y, S,

T, D, E, R, K, H, insertion, deletion,

Position 179 to G, A, V, L, I, W, P, M, F, N, Q, Y, S,

T, D, E, R, K, H, insertion, deletion, Position 190 to G, A, V, L, I, W, C, M, F, N, Q, Y, S,

T, R, K, H, deletion,

Position 193 to G, V, L, I, W, M, F, N, Q, Y, S, T, D,

E, R, H, deletion,

Position 196 to G, A, V, L, I, , P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,

Position 208 to A, V, L, I, W, C, M, F, Y, T, R, K, H,

Position 213 to N, oN, E,

Position 215 to A, L, I, W, M, F, Y,

Position 232 to A, insertion, Position 233 to L, insertion,

Position 234 to I, insertion,

Position 246 to insertion,

Position 250 to G, A, V, L, I, W, P, M, F, N, Q, Y, S,

T, D, E, R, H, insertion, deletion, Position 254 to G, V, L, I, W, M, F, N, Q, Y, S, D, E,

R, H, insertion, deletion,

Position 267 to G, A, V, L, I, , M, F, N, Q, Y, S, T,

D, E, R, H, insertion, deletion, Position 268 to G, V, L, I, W, C, M, N, Q, Y, S, T, D,

E, R, K, H, insertion, deletion,

Position 269 to G, A, V, L, I, , M, F, N, Q, Y, S, T, E, R, H, insertion, deletion,

Position 273 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,

87. The savinase-like subtilisin according to claim 86, wherein the subtilisin has at least 81%, preferably at least 96%, more preferably at least 98%, most preferably at least 99% homology to SEQ ID NO 24.

88. The savinase-like subtilisin according to claim 87, wherein the subtilisin has any of the amino acid sequence of SEQ ID NO 24, 26, 27, 28, 29, 30, 31, 32, 34, 35.

89. The protein variant according to claims 76-81 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO 25 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 25:

Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion,

Position 27 to G, L, I, , P, M, F, Y, T, H, Position 50 to G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion,

Position 52 to V, L, I, W, M, F, Y, S, T, R, deletion, insertion, Position 55 to G, A, V, L, I, , C, M, F, N, Q, Y, T,

D, E, R, K, H, deletion, insertion, Position 129 to ,

Position 133 to A, L, I, W, M, F, Y, R, Position 172 to G, A, V, L, I, S, T, H, deletion,

Position 186 to G, A, V, L, W, , F, N, Q, Y, S, T, D,

E, R, H, deletion,

Position 194 to W, N, Q, Y, D, H,

Position 195 to W, P, Y, deletion, Position 197 to G, V, L, I, , P, M, F, Q, Y, S, T, H, insertion, deletion,

Position 242 to insertion,

Position 249 to L, W, P, F, S, D, E, H, insertion, deletion, Position 252 to G, A, W, P, N, Q, Y, T, E, R, H, insertion, deletion,

Position 254 to G, V, L, I, W, M, F, N, Q, Y, S, D, E,

R, H, insertion, deletion,

Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion, deletion,

Position 260 to L, I, W, M, F, Y, T, H, insertion, deletion,

Position 265 to V, L, I, W, M, F, Y, insertion, deletion,

with the proviso that the amino acids of the parent enzyme are substituted to another amino acid.

90. The protein variant according to claims 76-81 having modi- fied immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO 10 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 10: Position 4 to V, L, W, M, F, Y, R,

Position 38 to V, L, I, W, M, F, N, Q, Y, T, H,

Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H,

Position 43 to G, L, H, Position 47 to G, A, V, L, I, , P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,

Position 49 to G, A, V, I, W, P, M, F, N, Q, Y, S, T,

D, E, R, H, insertion, deletion,

Position 54 to V, L, I, W, M, F, S, R, deletion, inser- tion,

Position 96 to L, W, F, Y, R, K, deletion,

Position 99 to V, L, I, W, M, F, Q, Y, H, deletion,

Position 113 to W, insertion,

Position 131 to W, Y, R, deletion, Position 133 to A, L, I, W, M, F, Y, R,

Position 137 to G, A, V, I, , P, M, N, Y, H, insertion, deletion,

Position 141 to G, V, L, I, , P, M, F, Q, S, D, E, H, insertion, deletion, Position 144 to L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,

Position 170 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, W, Y;

Position 173 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, H, deletion,

Position 181 to G, A, V, L, I, W, C, M, F, Q, Y, T, D,

R, K, H, deletion,

Position 185 to G, A, V, L, I, W, C, M, F, N, Q, Y, T,

E, H, deletion, Position 186 to G, A, V, L, , M, F, N, Q, Y, S, T, D, E, R, H, deletion,

Position 188 to G, A, V, L, , F, S, R, K, deletion,

Position 194 to , N, Q, Y, D, H,

Position 203 to V, F, Y, R, H, Position 210 to W, F, Y,

Position 211 to L, W, M, F, Y, H,

Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T,

D, E, K, H, insertion, deletion, Position 261 to L, N, S, H, insertion, deletion,

Position 262 to G, A, V, L, I, , P, F, N, Q, Y, T, D,

E, R, H, insertion, deletion,

Position 265 to V, L, I, W, M, F, Y, insertion, deletion,

91. The protein variant according to claims 76-81 having modi- fied immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO 11 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 11:

Position 38 to V, L, I, W, M, F, N, Q, Y, T, H,

Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H,

Position 45 to G, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,

Position 47 to G, A, V, L, I, , P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,

Position 49 to G, A, V, I, W, P, M, F, N, Q, Y, S, T,

D, E, R, H, insertion, deletion,

Position 50 to G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion, Position 52 to V, L, I, W, M, F, Y, S, T, R, deletion, insertion,

Position 53 to A, V, L, I, W, M, F, N, Q, Y, S, D, E, H, deletion, insertion,

Position 56 to G, V, L, I, , M, F, N, Q, Y, S, T, H, Position 58 to L, W, M, F, N, Y, R, insertion, dele- tion,

Position 96 to L, W, F, Y, R, K, deletion,

Position 97 to V, L, , C, , F, Y, H,

Position 98 to A, deletion,

Position 105 to insertion,

Position 109 to N, insertion,

Position 113 to W, insertion,

Position 115 to I, insertion,

Position 133 to A, L, I, W, M, F, Y, R,

Position 136 to G, A, W, P, N, Y, S, T, D, E, H, inser- tion, delet :ion,

Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion,

Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion,

Position 158 to V, L, I, W, M, F, Y,

Position 159 to A, W, M, Y, T, R, H,

Position 172 to G, A, V, L, I, S, T, H, deletion,

Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D,

E, R, H, deletion /

Position 189 to W, F, deletion,

Position 192 to G, L, I, , M, N, Q, Y, S, T, D, R, H, deletion,

Position 195 to W, P, Y, deletion,

Position 197 to G, V, L, I, , P, M, F, Q, Y, S, T, H, insertion, deletion, Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion, deletion, Position 261 to L, N, S, H, insertion, deletion, Position 265 to V, L, I, W, M, F, Y, insertion, dele- tion, with the proviso that the amino acids of the parent enzyme are substituted to another amino acid.

92. The protein variant according to claims 76-81 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO 33 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 33:

Position -6 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, Y, insertion, deletion,

Position -5 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, T, V, W, Y, insertion, deletion, Position -4 to A, C, D, E, F, G, H, I, K, L, M, N, Q,

R, S, T, V, W, Y, insertion, deletion,

Position -2 to A, C, E, F, G, H, I, K, L, , N, P, Q,

R, S, T, V, W, Y, insertion, deletion,

Position -1 to G, V, L, I, W, C, M, F, N, Q, Y, S, T, D, E, R, H, deletion,

Position 1 to V, L, I, W, M, F, Y, S, T, R,

Position 2 to G, V, I, M, F, N, Q, Y, S, T, H,

Position 3a to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, W, Y, insertion, deletion, Position 5 to V, L, I, W, M, F, N, Q, Y, T, R, H,

Position 6 to G, V, L, I, W, P, M, N, Q, T, D, E, R,

H,

Position 7 to G, A, V, L, I, W, P, M, F, N, Q, Y, S,

T, D, E, R, H, Position 8 to G, A, L, , P, C, M, F, N, Q, Y, S, T,

D, E, R, K, H,

Position 10 to G, A, V, I, W, P, M, N, Q, Y, S, T, D,

E, R, insertion, deletion, Position 12 to G, A, V, L, I, W, M, F, N, Q, Y, S, T,

D, E, insertion, deletion,

Position 13 to G, L, I, , P, M, F, N, Q, Y, S, D, E, H, insertion, deletion, Position 14 to V, L, I, W, P, M, F, N, Q, Y, T, R, H, insertion, deletion,

Position 15 to G, A, V, L, I, , P, M, F, N, Q, Y, S, T, E, H, insertion, deletion,

Position 16 to G, A, V, L, I, , P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,

Position 17 to G, A, V, I, , P, M, F, Y, H, insertion, deletion,

Position 18 to G, A, L, I, W, P, M, F, N, Q, Y, T, D,

E, H, insertion, deletion, Position 19 to A, V, I, W, M, F, N, Y, S, T, D, R, H, insertion, deletion,

Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion,

Position 22 to G, V, L, I, W, M, F, Y, S, T, insertion, deletion,

Position 23 to G, A, V, L, I, W, M, F, Y, E, R, H, insertion, deletion,

Position 24 to G, V, L, I, W, M, F, N, Q, Y, S, D, E,

R, insertion, deletion, Position 25 to G, A, V, L, I, W, M, F, N, Q, Y, S, T,

D, E, R, H, insertion, deletion,

Position 26 to G, A, V, L, I, W, M, F, N, Q, Y, S, T,

D, E, R, H, insertion, deletion,

Position 27 to G, L, I, , P, M, F, Y, T, H, Position 28 to G, A, V, L, I, , P, M, F, N, Q, Y, S,

T, D, E, R, K, H, insertion, deletion,

Position 28a to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, W, Y, insertion, deletion, Position 29 to G, A, V, L, I, W, P, M, F, N, Q, Y, S,

T, D, E, R, K, H, insertion, deletion,

Position 33 to V, L, I, , C, M, F, N, Q, Y, R, H,

Position 35 to G, A, V, L, I, , M, F, N, Q, Y, S, T, D, E, R, K, H, deletion,

Position 37 to L, I, W, M, F, N, Q, Y, S, R, H,

Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H,

Position 42 to G, A, L, , C, M, F, N, Q, Y, S, T, D, E , R, H, Position 43 to G, L, H,

Position 44 to G, V, L, I, , P, M, F, Y, S, T,

Position 44a to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, , Y, insertion, deletion,

Position 44b to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion,

Position 46 to G, A, L, I, W, P, , F, Y, H, insertion, deletion,

Position 48 to A, L, I, P, M, F, N, Y, D, H, insertion, deletion, Position 51 to ^' V, L, I, W, M, F, N, Y, R, deletion, insertion,

Position 52 to V, L, I, W, M, F, Y, S, T, R, deletion, insertion,

Position 53 to A, V, L, I, W, M, F, N, Q, Y, S, D, E, H, deletion, insertion,

Position 55 to G, A, V, L, I, W, C, M, F, N, Q, Y, T,

D, E, R, K, H, deletion, insertion,

Position 56 to G, V, L, I, W, M, F, N, Q, Y, S, T, H,

Position 57 to G, A, V, L, I, , M, F, N, Q, Y, S, T, D, E, R, K, H,

Position 58 to L, W, M, F, N, Y, R, insertion, deletion,

Position 61 to V, L, I, W, M, F, Y, insertion, deletion, Position 64 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion, Position 75 to L,

Position 81 to insertion, Position 86 to G, A, V, L, I, , M, F, N, Q, Y, T, D,

E, R, H,

Position 87 to A, V, L, I, W, M, F, Q, Y, S, T, D, E,

H,

Position 88 to A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H,

Position 89 to G, V, L, I, W, P, F, N, Y, T, E,

Position 91 to G, A, V, L, I, W, P, M, N, Y, S, T, D, E, R, H, insertion, deletion,

Position 92 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,

Position 94 to G, V, L, I, W, P, M, F, N, Y, T, D, E,

K, H, insertion, deletion,

Position 96 to L, , F, Y, R, K, deletion,

Position 97 to V, L, W, C, M, F, Y, H, Position 98 to deletion,

Position 101 to V, I, W, M, F, N, Q, Y, H,

Position 102 to V, L, I, W, M, F, Y, R, H, G, deletion,

Position 108 to I, insertion,

Position 109 to N, insertion, Position 111 to insertion,

Position 112 to E, insertion,

Position 113 to , insertion,

Position 114 to insertion,

Position 115 to I, insertion, Position 117 to N, insertion,

Position 118 to N, insertion,

Position 119 to M, insertion,

Position 127 to G, A, V, I, , M, F, Y, R, H, L,

Position 133 to A, L, I, W, M, F, Y, R, Position 134 to L, I, , F, N, Q, Y, R, H, insertion, deletion,

Position 135 to G, L, P, C, N, Q, T, R, H,

Position 136 to G, A, W, P, N, Y, S, T, D, E, H, inser- tion, deletion,

Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion,

Position 138 to G, A, V, L, I, , P, M, F, N, Q, Y, S,

T, D, E, R, H, insertion, deletion, Position 139 to G, A, V, L, I, , P, C, M, F, N, Q, Y,

S, T, D, E, R, K, H, insertion, deletion,

Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion,

Position 142 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,

Position 144 to L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,

Position 145 to G, V, L, I, W, M, F, Q, Y, D, E, R, H, insertion, deletion, Position 146 to G, A, W, L, I, , M, F, N, Q, Y, T, D,

E, R, H, insertion, deletion,

Position 147 to G, A, V, L, , M, F, N, Q, Y, S, T, D,

E, R, K, H, insertion, deletion,

Position 148 to G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,

Position 156 to V, I, W, F, R,

Position 158 to V, L, I, W, M, F, Y,

Position 160 to W, M, F, Y, R, H,

Position 161 to I, W, M, F, Y, H, Position 162 to I, W, F, Y, R,

Position 163 to V, W, M, F, H,

Position 167 to R, K,

Position 169 to C, E, F, G, H, I, K, L, M, N, Q, R, T,

V, W, Y, Position 170 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, W, Y, insertion, deletion,

Position 171 to D, deletion,

Position 174 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,

Position 176 to G, A, V, L, I, W, P, C, M, F, N, Q, Y,

S, T, D, E, R, K, H, insertion, deletion,

Position 182 to A, V, L, I, , C, M, F, N, Q, Y, S, T,

D, E, H, deletion, Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D,

E, R, H, deletion,

Position 188 to G, A, V, L, W, F, S, R, K, deletion,

Position 191 to A, V, L, I, W, M, F, Y, T, R, H, dele- tion, Position 192 to G, L, I, W, , N, Q, Y, S, T, D, R, H, deletion,

Position 193 to G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,

Position 194 to W, N, Q, Y, D, H, Position 195 to W, P, Y, deletion,

Position 196 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,

Position 197 to G, V, L, I, , P, M, F, Q, Y, S, T, H, insertion, deletion, Position 198 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,

Position 203 to V, F, Y, R, H,

Position 205 to W, F, Y, R, K,

Position 215 to A, L, I, W, M, F, Y, Position 216 to A, L, I, W, M, F, Y, R,

Position 217 to , R,

Position 219 to G, A, V, L, I, W, F, Y, R, H,

Position 233 to insertion,

Position 234 to I, insertion, Position 236 to insertion,

Position 237 to insertion,

Position 238 to insertion,

Position 239 to insertion, Position 240 to insertion,

Position 243 to insertion,

Position 246 to insertion,

Position 247 to G, V, I, W, P, F, Y, S, T, R, insertion, deletion, Position 249 to L, W, P, F, S, D, E, H, insertion, deletion,

Position 252 to G, A, , P, N, Q, Y, T, E, R, H, insertion, deletion,

Position 254 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,

Position 262 to .G, A, V, L, I, W, P, F, N, Q, Y, T, D,

E, R, H, insertion, deletion,

Position 264a to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, W, Y, insertion, deletion, Position 270 to G, L, I, W, P, M, F, N, Q, Y, S, T, D,

E, R, K, H, insertion, deletion,

Position 273 to G, A, V, L, I, , P, M, F, N, Q, Y, S,

T, D, E, R, K, H, insertion, deletion,

Position 274 to , P, M, F, N, Q, Y, T, D, E, R, H, Position 275 to G, A, V, L, I, , M, F, N, Y, T, D, insertion, deletion,

Position 276 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, V, W, Y, insertion, deletion,

93. The protein variant according to claims 76-81 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO 33 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 33 :

Position 5 to V, L, I, W, M, F, N, Q, Y, T, R, H,

Position 22 to G, V, L, I, , M, F, Y, S, T, insertion, deletion,

Position 26 to G, A, V, L, I, W, M, F, N, Q, Y, S, T,

D, E, R, H, insertion, deletion, Position 28 to G, A, V, L, I, W, P, M, F, N, Q, Y, S,

T, D, E, R, K, H, insertion, deletion,

Position 37 to L, I, W, M, F, N, Q, Y, S, R, H,

Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H,

Position 44 to G, V, L, I, , P, M, F, Y, S, T, Position 51 to V, L, I, W, M, F, N, Y, R, deletion, in- sertion,

Position 52 to V, L, I, W, M, F, Y, S, T, R, deletion, insertion,

Position 55 to G, A, V, L, I, , C, M, F, N, Q, Y, T, D, E, R, K, H, deletion, insertion,

Position 58 to L, W, M, F, N, Y, R, insertion, dele- tion,

Position 61 to V, L, I, W, M, F, Y, insertion, dele- tion,

Position 64 to G, V, L, I, W, P, C, M, F, N, Q, Y, S,

T, D, E, R, K, H, in tion, deletion,

Position 87 to A, V, L, I, W, M, F, Q, Y, S, T, D, E,

H,

Position 97 to V, L, W, C, M, F, Y, H,

Position 98 to deletion,

Position 101 to V, I, W, M, F, N, Q, Y, H,

Position 102 to V, L, I, W, M, F, Y, R, H, G, deletion,

Position 109 to N, insertion,

Position 112 to E, insertion, Position 118 to N, insertion,

Position 127 to G, A, V, I, W, M, F, Y, R, H, L,

Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion, Position 146 to G, A, W, L, I, , M, F, N, Q, Y,^' T, D,

E , R, H, inserti_'on, letion,

Position 156 to V, I, W, F, R,

Position 158 to V, L, I, W, M, F, Y,

Position 161 to I, W, M, F, Y, H, Position 188 to G, A, V, L, W, F, S, R, K, deletion,

Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion,

Position 194 to , N, Q, Y, D, H,

Position 195 to W, P, Y, deletion, Position 203 to V, F, Y, R, H,

Position 216 to A, L, I, W, M, F, Y, R,

Position 236 to insertion,

Position 237 to insertion,

Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion, deletion,

Position 264a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, , Y, insertion, deletion,

with the provisio that the amino acids of the parent enzyme are substituted to another mino acid.

94. The protein variant according to claim 76, wherein the lipolytic enzyme comprises one or more of the following substi- tutions corresponding to any of the following in SEQ ID NO: 1:

Q15 to A, C, D, E, F, G, I, K, L, M, N, P, R, S, T, V, , Y; Y16 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W; A18 to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; A19 to C, D, E, F, G, H, I, K, L, M, N, Q, R, S, V, , Y;

A20 to C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, , Y;

N25 to A ¥ , w, Y;

N26 to A w , Y; E43 to A Y

V44 to A r Y;

K46 to A Y

A47 to C w Y;

A49 to C Y L52 to A ¥ W, Y;

Y53 to A ¥ ;

S54 to A W Y;

G65 to A ¥ _r W, Y;

L67 to A Y; A68 to C ¥ , Y;

L69 to A W Y;

T72 to A W Y;

K74 to A ¥ W, Y;

L75 to A ¥ , Y; V77 to A T , Y;

S79 to A ¥ , Y;

R81 to A w, Y;

S83 to A Y,

S85 to A W89 to A ¥, Y;

L97 to A

K98 to A

E99 to C

G106 to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, Y; C107 to A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;

R108 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, Y;

G109 to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, , Y;

T123 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, Y;

L124 to A, C, D, E, F, G, H, I, K, M, N, P, Q, R, T, V, , Y; K127 Y; E129 Y; A131 W, Y; ¥132 Y; Y138 ¥, ; ¥140 Y; L147 W, Y A150 W, Y T153 W, Y Y164 ¥, W D165 D167 S170 Y Y171 ¥ W; G172 W Y; A173 Y P174 W Y; R175 Y ¥176 w, Y; G177 W Y; R179 Y; A182 W Y; Y194 ¥, W; H198 w, Y; N200 Y P207 W Y; P208 W Y; R209 G212 W Y; S214 W Y; H215 W Y; S216 Y S217 Y; P218 W Y; E219 to C , Y;

Y220 to I, K, L, M, N, P, Q, R, S, T, ¥, ;

K223 to I, L, M, N, Q, S, T, ¥, W, Y;

S224 to I, K, L, M, N, Q, T, ¥, , Y; D234 to C

1235 to K, L, M, N, P, Q, R, S, T, ¥, W, Y;

K237 to I, L, N, P, Q, S, T, ¥, , Y;

1238 to K, L, M, N, P, Q, R, S, T, ¥, W, Y;

D242 to C P, , Y; A243 to C K, L, M, N, P, Q, R, S, ¥, W, Y;

P250 to A I, K, L, M, N, Q, R, S, T, ¥, , Y;

P253 to A I, K, L, M, N, Q, S, T, ¥, W, Y;

D254 to C Y;

1255 to C N, Q, W, Y; P256 to C L, M, N, Q, R, ¥, W, Y;

Y261 to A M, N, P, Q, R, S, T, ¥.

95. The protein variant according to claim 94, wherein the lipolytic enzyme comprises one or more of the substitutions corresponding to any of the following in SEQ ID NO: 1:

G65 to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y;

L67 to A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, ¥, , Y; R81 to A, D, E, F, G,^' H, I, K, L, M, N, P, Q, S, T, ¥, W, Y;

S83 to A, C' D, E, F, G, H, I, K, L, M, N, Q, R, ¥, W, Y;

S85 to A, D, E, G, H, I, L, M, N, Q, ¥, W, Y;

L97 to A, C, D, E, F, G, H, I, K, N, P, R, S, T, , Y;

L124 to A, C, D, E, F, G, H, I, K, M, N, P, Q, R, T, ¥, W, Y; E129 to A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, ¥, W, Y;

Y164 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W

R179 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, ¥, , Y

A182 to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y

P207 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, ¥, W, Y P208 to A, c, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, ¥, W, Y; R209 to C, D, F, G, H, I, K, L, M, N, Q, T, ¥, W, Y; G212 to A, c, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y S214 to A, c, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, ¥, , Y H215 to A, c, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, ¥, , Y S216 to A, c, D, E, F, G, H, I, K, L, M, N, Q, R, T, ¥, W, Y; S217 to A, c, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, ¥, W, Y; P218 to A, c, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, ¥, , Y; E219 to C, D, F, H, I, M, P, w, Y; Y220 to A, c, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, ; A243 to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, ¥, , Y; P250 to A, G, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, ¥, W, Y; P253 to A, c, D, E, F, G, H, I, K, L, M, N, Q, S, T, ¥, , Y;

96. The protein variant according to claim 95, wherein the lipolytic comprises one or more of the following substitutions:

P207 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, ¥, , Y P208 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, ¥, W, Y

S214 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, ¥, W, Y

5216 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, ¥, W, Y;

5217 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, ¥, W, Y; A243 to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, ¥, W, Y; P250 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, ¥, W, Y;

P253 to A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T, ¥, W, Y;

97. The protein variant according to claim 94-96, wherein the parent lipolytic enzyme has at least 80% homology with SEQ ID N0.1.

98. The protein variant according to claim 76, wherein the car- bohydrase is a glucoamylase comprising one or more of the fol- lowing substitutions corresponding to any of the following in SEQ ID NO 36:

Position 68 to A, C, D, E, F, G, H, I, K, L, M, N, P, 5 Q, R, S, T, ¥, W, Y, deletion, insertion, Position 94 to insertion,

Position 102 to insertion,

Position 122 to insertion,

Position 125 to insertion, o Position 272 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y, deletion, insertion,

Position 345 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, ¥, , Y, deletion, insertion,

Position 348 to A, C, D, E, F, G, H, I, K, L, M, N, P, s Q, R, S, T, ¥, , Y, deletion, insertion, Position 353 to insertion,

Position 357 to insertion,

Position 359 to insertion,

Position 450 to insertion, 0 Position 451 to insertion,

Position 468 to insertion,

with the proviso that the amino acids of the parent enzyme are substituted to another amino acid. 5

99. The protein variant according to claims 98, wherein the enzyme is at least 81 % homologous, preferably 90% homologous, more preferably 95% homologous, most preferably 99% homologous to Carezyme core (SEQ ID NO 36) . 0

100. The protein variant according to claim 76, wherein the car- bohydrase is a Thermamyl-like α-amylase comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO 2 : Position TYR 8 to A, C, D, G, K, M, P, R, W,

Y, insertion;

Position ASP 25 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y, insertion;

Position ASP 26 to A, C, D, E, F, G, H, I,

K, L, M, P, Q, R, S, T, ¥, W, Y;

Position ALA 27 to C, D, E, F, G, H, I, K,

L, M, N, P, Q, R, S, T, ¥, W, Y; Position SER 28 to A, C, D, F, G, H, I, K,

L, M, P, Q, R, S, T, ¥, W, Y;

Position ASN 29 to A, C, D, G, K, M, P, R,

W, Y;

Position ARG 31 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, ¥, W, Y;

Position PRO 41 to C, D, E, F, G, H, I, K,

L, , N, P, Q, R, S, T, ¥, W, Y, deletion, insertion;

Position PRO 42 to C, D, E, F, G, H, I, K,

L, M, N, P, Q, R, S, T, ¥, W, Y, deletion, insertion; Position TYR 54 to A, C, D, E, G, K, M, P,

R, Y, insertion;

Position TYR 57 to A, C, D, G, K, M, P, R,

W, Y, insertion;

Position LEU 62 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y, deletion, insertion;

Position GLY 63 to A, C, D, E, F, G, H, I,

K, L, M, N, P, Q, R, S, T, ¥, , Y, deletion, insertion;

Position GLY 76 to A, C, D, E, F, G, H, I,

K, L, M, N, P, Q, R, S, T, ¥, W, Y, deletion, insertion; Position ARG 78 to A, C, D, E, F, G, H, I,

K, L, M, N, P, Q, R, S, T, ¥, W, Y, deletion, insertion;

Position SER 79 to A, C, D, E, F, G, H, I,

K, L, M, N, P, Q, R, S, T, ¥, , Y, deletion, insertion; Position LEU 88 to A, C, D, E, F, G, H, I,

K, L, M, N, P, Q, R, S, T, ¥, W, Y, deletion, insertion;

Position GLY 92 to A, C, D, E, F, G, H, I,

K, L, M, N, P, Q, R, S, T, ¥, , Y, deletion, insertion; Position ASN 102 to A, C, E, F, G, H, I, L,

M, P, Q, S, T, ¥, W, Y, insertion;

Position ALA 107 to A, C, D, E, F, G, H, I,

K, L, M, N, P, Q, R, S, T, ¥, W, Y, deletion, insertion;

Position ASP 108 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y, deletion, insertion;

Position ALA 109 to A, C, D, E, F, G, H, K,

M, N, P, Q, R, S, W, Y;

Position LYS 138 to A, C, E, F, G, I, L, M,

N, P, Q, S, T, ¥, W, insertion; Position ASP 140 to A, C, E, F, G, I, K, L, position PRO 142 to C, D, E, F, G, H, I, K,

L, M, N, Q, R, S, T, ¥, W, Y;

Position ARG 144 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, Y;

Position GLN 170 to A, C, E, F, G, H, I, K,

L, M, N, P, Q, R, S, T, ¥, W, Y;

Position ILE 173 to A, C, D, E, F, G, H, I,

K, L, M, N, P, Q, R, S, T, ¥, W, Y; Position ASP 195 to A, C, D, E, F, G, H, I,

K, L, M, P, Q, R, S, T, ¥, W, Y, deletion, insertion;

Position TYR 196 to A, C, D, G, K, M, P, R,

W, Y, insertion;

Position ASP 232 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y, deletion, insertion;

Position ALA 233 to A, C, D, E, I, K, L, M,

N, P, Q, R, , Y, deletion, insertion;

Position GLN 331 to A, C, D, F, G, H, I, K,

M, N, P, Q, R, S, T, ¥, , Y, deletion, insertion; Position TYR 349 to A, C, D, G, K, M, P, R,

W, Y, insertion;

Position ILE 352 to A, C, D, E, F, G, H, I,

K, L, M, N, P, Q, R, S, T, ¥, , Y, deletion, insertion; Position GLN 357 to C, D, E, G, H, I, K, L,

M, N, P, Q, R, S, T, ¥, W, Y, deletion, insertion;

Position ASP 366 to A, C, D, E, F, G, H, I,

K, L, M, P, Q, R, S, T, ¥, W, Y, deletion, insertion;

Position TYR 367 to C, E, F, H, K, M, N, P, Q, R, ¥, , insertion;

Position TYR 368 to A, C, D, G, K, M, P, R,

W, Y, insertion;

Position ILE 370 to A, C, D, E, F, G, H, I,

K, L, M, N, P, Q, R, S, T, ¥, , Y; Position ALA 380 to A, C, D, E, F, G, H, I,

K, L, M, N, P, Q, R, T, ¥, W, Y;

Position LYS 381 to A, C, D, E, F, G, H, I,

K, L, M, N, P, Q, S, T, ¥, W, Y, deletion, insertion;

Position ILE 382 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y, deletion, insertion;

Position PRO 384 to C, D, E, F, G, H, I, K,

M, N, P, Q, R, S, T, ¥, W, deletion, insertion;

Position LEU 386 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y, deletion, insertion;

Position ARG 389 to A, C, D, E, F, G, H, I,

K, L, M, N, P, Q, R, S, T, ¥, W, Y, deletion, insertion;

Position GLN 390 to A, C, D, E, F, G, H, I,

L, M, N, P, Q, S, T, ¥, W, Y;

101. The protein variant according to claim 100, wherein the amylase comprises one or more of the following substitutions:

Position PRO 41 to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y, deletion, insertion;

Position PRO 42 to C, D, E, F, G, H, I,

K, L, M, N, P, Q, R, S, T, ¥, , Y, deletion, insertion;

Position ALA 109 to A, C, D, E, F, G, H,

K, M, N, P, Q, R, S, , Y; Position LYS 138 to A, C, E, F, G, I, L,

M, N, P, Q, S, T, ¥, W;

Position ASP 140 to A, C, E, F, G, I, K,

L, M, N, P, Q, S, T, ¥, ;

Position PRO 142 to C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, ¥, , Y;

Position ARG 144 to A, C, D, E, F, G, H,

I, K, L, M, N, P, Q, R, S, T, ¥, Y;

Position ASP 366 to A, C, D, E, F, G, H,

I, K, L, M, P, Q, R, S, T, ¥, W, Y, deletion, insertion; Position TYR 367 to C, E, F, H, K, M, N,

P, Q, R, ¥, , insertion;

Position TYR 368 to A, C, D, G, K, M, P,

R, , Y, insertion;

Position ALA 380 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, ¥, W, Y;

Position LYS 381 to A, C, D, E, F, G, H,

I, K, L, M, N, P, Q, S, T, ¥, W, Y, deletion, insertion;

Position ILE 382 to A, C, D, E, F, G, H,

I, K, L, M, N, P, Q, R, S, T, ¥, , Y, deletion, insertion;

Position PRO 384 to C, D, E, F, G, H, I,

K, M, N, P, Q, R, S, T, ¥, , deletion, insertion;

Position ARG 389 to A, C, D, E, F, G, H,

I, K, L, M, N, P, Q, R, S, T, ¥, , Y, deletion, insertion; with the proviso that the amino acids of the parent enzyme are substituted to another amino acid.

5

102. The protein variant according to claims 100-101, wherein the enzyme is at least 81 % homologous, preferably 90% homologous, more preferably 95% homologous, most preferably 99% homologous to SEQ ID NO 2.

10

103. The protein variant according to claims 100-102, wherein the enzyme has any of the amino acid sequence of SEQ ID NO 2 , 4 , 5, 37.

15 104. A cellulase variant of a microbial parent cellulase having a catalytically active domain classified in family 45, said variant comprises a substitution of one or more amino acid residues at a position corresponding to a position in SEQ ID NO: 4 from the group consisting of:

20

Position 1 to C, D, E, F, G, H, I, K, L, M, N, P, Q,

R, S, T, ¥, W, Y;

Position 2 to A, C, E, F, G, H, I, K, L, M, P, Q, R,

S, T, ¥, , Y; 25 Position 7 to A, C, D, E, F, G, H, K, L, M, N, P, Q,

S, T, ¥, Y;

Position 20 to C, D, F, H, I, L, M, N, P, Q, S, T, ¥, , Y;

Position 23 to A, C, D, E, F, G, H, I, K, L, M, N, Q, 30 R, S, T, ¥, , Y;

Position 27 to A, C, D, E, F, G, H, I, K, L, M, N, Q,

R, S, T, ¥, , Y;

Position 29 to A, C, D, E, G, H, I, K, L, M, N, P, Q,

R, S, T, ¥, , Y; Position 36 to A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, ¥, , Y;

Position 37 to C, D, E, F, G, H, I, K, L, M, P, Q, T, ¥, , Y; Position 38 to A, C, D, E, G, H, K, M, N, P, R, S, T, V, W, Y;

Position 40 to A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y;

Position 41 to A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y;

Position 44 to A, C, D, E, F, H, I, L, M, N, S, T, W,

Y;

Position 54 to A, C, D, E, G, H, I, K, L, M, N, P, Q,

R, S, T, ¥, W; Position 59 to A, C, D, E, F, G, H, I, K, L, M, N, P,

R, S, T, ¥, , Y;

Position 61 to A, C, D, E, F, G, H, I, K, L, M, N, Q,

R, S, T, ¥, , Y;

Position 62 to A, C, D, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, Y;

Position 83 to C, D, E, F, G, H, I, K, L, M, N, P, Q,

R, S, T, ¥, W, Y;

Position 84 to A, C, D, E, F, H, I, K, L, M, N, P, Q,

R, S, T, ¥, W, Y; Position 95 to A, C, D, F, G, H, I, K, L, M, N, P, Q,

R, S, ¥, , Y;

Position 96 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, ¥, W, Y;

Position 97 to C, D, E, F, H, I, K, L, M, N, P, Q, R, S, ¥, W, Y;

Position 98 to C, D, E, F, G, H, I, K, L, M, N, Q, R,

S, T, ¥, W, Y;

Position 100 to C, D, E, F, G, H, I, K, L, M, N, P, Q,

S, T, ¥, W, Y; Position 101 to A, C, D, E, F, H, I, K, L, M, N, P, Q,

R, S, T, ¥, , Y;

Position 131 to C, D, E, F, K, M, P, R, S, W, Y;

Position 133 to A, C, E, F, G, H, I, L, M, P, R, S, T, ¥, W, Y;

Position 134 to C, D, E, F, H, I, K, L, M, N, P, Q, R,

S, T, ¥, W, Y;

Position 136 to A, C, E, F, G, H, I, K, L, M, N, P, Q,

R, S, ¥, , Y; Position 142 to A, C, E, F, G, H, I, K, M, N, P, Q, R, ¥, W, Y;

Position 143 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T,

V, W, Y;

Position 145 to C, E, F, G, H, I, K, L, M, P, R, S, T, ¥, W, Y;

Position 146 to A, C, D, F, G, H, I, K, L, M, N, P, T, ¥, W, Y;

Position 151 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T,

V, , Y;

Position 153 to C, D, E, F, G, H, I, M, N, P, Q, S, T,

V, , Y; Position 154 to A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T,

V, W, Y;

Position 155 to A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T,

V, W, Y;

Position 157 to A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y;

Position 158 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T,

V, , Y;

Position 160 to A, C, D, E, F, G, H, I, K, L, M, N, Q,

R, S, T, ¥, W, Y; Position 162 to C, D, E, F, G, H, I, K, L, M, N, Q, R,

S, T, ¥, , Y;

Position 163 to A, C, D, E, F, G, H, I, K, M, P, Q, R,

S, T, Y; Position 164 to A, C, D, E, F, G, H, I, L, M, N, P, Q,

R, S, T, ¥, , Y;

Position 165 to A, C, D, E, F, G, H, I, K, L, M, N, Q,

R, S, T, ¥, , Y;

Position 168 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, S, T, ¥, W;

Position 169 to A, C, D, E, G, H, I, K, L, M, N, P, Q,

R, S, T, ¥, Y;

Position 170 to A, C, D, E, G, H, I, K, L, M, N, P, Q,

S, T, ¥, , Y;

Position 174 to A, C, D, E, G, H, I, K, L, N, P, Q, R,

S, T, ¥, Y;

Position 176 to A, C, E, F, G, H, I, K, L, M, P, Q, R,

S, T, ¥, , Y;

Position 177 to C, D, E, F, G, H, I, K, L, M, P, Q, R,

S, T, ¥, W, Y;

Position 178 to A, C, E, F, G, H, I, K, L, M, Q, R, S,

T, V, W, Y;

Position 180 to A, C, D, E, F, G, H, I, K, M, N, Q, R,

S, T, ¥, , Y;

Position 183 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, T, ¥, , Y;

Position 191 to C, D, E, F, G, H, I, K, L, M, N, P, Q,

R, S, T, ¥, W, Y;

Position 195 to C, D, E, F, G, H, I, K, L, M, N, P, Q, , , 1 , ¥, ,

Position 197 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, R, V, W, Y; and

Position 200 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, S, T, V, W, Y;

105. The protein variant according to claim 104, wherein the carbohydrase comprises one or more of the following substitutions :

Position 20 to C, D, F, H, I, L, M, N, P, Q, S, T, ¥, , Y;

Position 23 to A, C, D, E, F, G, H, I, K, L, M, N, Q,

R, S, T, ¥, , Y;

Position 27 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, ¥, , Y;

Position 83 to C, D, E, F, G, H, I, K, L, M, N, P, Q,

R, S, T, ¥, , Y;

Position 84 to A, C, D, E, F, H, I, K, L, M, N, P, Q,

R, S, T, ¥, W, Y; Position 95 to A, C, D, F, G, H, I, K, L, M, N, P, Q,

R, S, ¥, W, Y;

Position 96 to A, C, D, E, F, G, H, I, K, L, M, N, P,

Q, V, W, Y;

Position 97 to C, D, E, F, H, I, K, L, M, N, P, Q, R, S, ¥, W, Y;

Position 98 to C, D, E, F, G, H, I, K, L, M, N, Q, R,

S, T, ¥, , Y;

Position ALA 100 to C, D, E, F, G, H, I, K,

L, M, N, P, Q, S, T, ¥, W, Y; and

Position 101 to A, C, D, E, F, H, I, K, L, M, N, P, Q,

R, S, T, ¥, , Y;

Position 131 to C, D, E, F, K, M, P, R, S, W, Y;

Position 142 to A, C, E, F, G, H, I, K, M, N, P, Q, R, V, W, Y; Position 143 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T,

¥, W, Y;

Position 145 to C, E, F, G, H, I, K, L, M, P, R, S, T, ¥, W, Y;

Position 151 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T,

¥, W, Y; Position 154 to A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y;

Position 155 to A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; 5 Position 157 to A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y;

Position 158 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, , Y;

10

106. The protein variant according to claims 104-105, wherein the enzyme is at least 81 % homologous, preferably 90% homologous, more preferably 95% homologous, most preferably 99% homologous to Carezyme core (SEQ ID NO 4) .

15

107. The protein variant according to claim 76, wherein the laccase is a Coprinus-like laccase.

20 108. The protein variant according to claim 107, wherein Laccase comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO 3 :

Position 5 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, 25 ¥, , Y;

Position 8 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,

¥, , Y;

Position 10 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y; 30 Position 12 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 22 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 23 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 30 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 39 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 40 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 41 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y;

Position 42 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 43 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 51 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 53 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 55 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y;

Position 58 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 59 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 60 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 71 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 72 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y;

Position 78 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 79 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 80 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 100 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 101 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 102 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 112 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y;

Position 113 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 114 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 118 to A, C, D, E, F, G, H, I, K, L, , N, P, Q, R, S,

T, ¥, W, Y;

Position 139 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 142 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y;

Position 155 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 157 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 165 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 166 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 168 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y;

Position 175 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 180 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 183 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 186 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 190 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 191 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 192 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y;

Position 193 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 211 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 213 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 231 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 234 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y;

Position 236 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 241 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 251 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 257 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 259 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y;

Position 265 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 275 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 286 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 294 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 295 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 296 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 299 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y;

Position 300 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, V, W, Y;

Position 301 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 302 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 306 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 313 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y;

Position 314 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 315 to A, C, D, E, F, G, H, I, K, L, , N, P, Q, R, S,

T, ¥, W, Y; Position 320 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 321 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 322 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y;

Position 324 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 329 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 332 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 335 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 336 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 339 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 344 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y;

Position 345 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 348 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 349 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 350 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 366 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y;

Position 367 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 369 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 370 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 371 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 372 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y;

Position 375 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 378 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 379 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 389 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y; Position 390 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 409 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 410 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y;

Position 414 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 416 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 418 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 419 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 420 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y;

Position 430 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 432 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 433 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 434 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 442 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y;

Position 443 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 445 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y; Position 446 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 469 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y; Position 473 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 485 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 488 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y;

Position 490 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 491 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 492 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 493 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 494 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y;

Position 495 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 496 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 499 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 500 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 501 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, , Y;

with the proviso that the amino acids of the parent protein is substituted to another amino acid.

109. The protein variant according to claim 108, wherein Laccase comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO 3 :

Position 59 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 96 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y; Position 100 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 181 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 348 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, ¥, W, Y;

Position 369 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, , Y;

Position 414 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y; Position 432 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

Position 493 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,

T, ¥, W, Y;

110. A subtilisin variant comprising one or more of the insertions, substitutions and/or deletions in any of the positions according to claims 77-93.

111. The variant according to claim 110, wherein the subtilisin has at least 60%, preferably at least 70%, more preferably at least 80, even more preferably at least 90, still more prefera- bly at least 95%, most preferably at least 99% homology to SEQ ID NO; 10.

112. A lipolytic enzyme comprising one or more of the inser- tions, substitutions and/or deletions in any of the positions according to claims 94-97.

113. A glycoamylase variant comprising one or more of the insertions, substitutions and/or deletions in any of the positions according to claims 98-99.

114. The variant according to claim 113, wherein the variant has at least 60%, preferably at least 70%, more preferably at least 80, even more preferably at least 90, still more preferably at least 95%, most preferably at least 99% homology to SEQ ID NO: 36.

115. A Thermamyl-like α-amylase comprising one or more of the insertions, substitutions and/or deletions in any of the posi- tions according to claims 100-103.

116. The variant according to claim 115, wherein the variant has at least 60%, preferably at least 70%, more preferably at least 80, even more preferably at least 90, still more preferably at least 95%, most preferably at least 99% homology to SEQ ID NO; 2.

117. A cellulase variant comprising one or more of the insertions, substitutions and/or deletions in any of the positions according to claims 104-106.

118. The variant according to claim 117, wherein the variant has at least 60%, preferably at least 70%, more preferably at least 80, even more preferably at least 90 , still more preferably at least 95%, most preferably at least 99% homology to SEQ ID NO; 10.

119. A coprinus-like laccase variant comprising one or more of 5 the insertions, substitutions and/or deletions in any of the positions according to claims 107-109.

120. A composition comprising a protein variant as defined in any of claims 22-119.

10

121. The composition according to claim 120, wherein the composition is in form of a pharmaceutical composition such as a vaccine.

15 122. The composition according to claim 120, wherein the compositions is in form of a industrial composition such as, a detergent composition, personal care composition.

123. The use of the composition as defined in claim 120 for the 20 production of a pharmaceutical.

124. The use of the composition as defined in claim 120 for industrial application.

25 125. A DNA construct comprising a DNA sequence encoding a protein variant as defined in any of claims 22-119.

126. An expression vector comprising a DNA construct according to claim 125.

30

127. A host cell which is capable of expressing a polypeptide and comprising a DNA construct as defined in claim 125.

128. A host cell which is capable of expressing a polypeptide and which is transformed by an expression vector according to claim 126.

5 129. A host according to claims 127-128, which is a fungal cell, an insect cell, a mammalian cell, or a plant cell.

130. A method of producing a protein variant having reduced immunogenicity as compared to the parent protein, comprising:

10

- culturing a host according to any of claims 127-129 in a suitable culture medium to obtain expression and secretion of the protein into the medium, followed by

is - isolation of the protein from the culture medium.

131. A kit for characterizing specificity of the allergic re- 20 sponse of a patient, comprising a set of antibody binding peptide sequences corresponding to at least one epitope on at least one potential allergen.

132. The kit according to claim 131, for which the antibody 25 binding sequences each are specific for one out of a known range of allergens, such that the characterization of allergic specificity becomes less susceptibility to cross-reactivity interferences .

30 133. A kit according to claims 131-132, which further comprises other diagnostic reagents, which facilitate determination of the serum response to each of the antibody binding sequences.

134. A kit according to claims 131-133, which further comprises allergen vaccines, which can be administered to the patient according to the test results obtained using the antibody binding sequences .