Methods for Purifying Pertussis Toxin and Peptides Useful Therefor

Abstract

The present invention relates to reagents and methods for purifying pertussis toxin (PT).

Description

RELATED APPLICATIONS

[0001] This application is a continuation U.S. Ser. No. 13/561,471 filed Jul. 30, 2012, which is a continuation of U.S. Ser. No. 10/579,655 filed Aug. 10, 2007, now U.S. Pat. No. 8,299,210B2 issued on Oct. 30, 2012, which is the National Stage Application under 35 U.S.C. §371 of International Application No. PCT/US2004/038700 filed Nov. 18, 2004, which claims priority to U.S. Provisional Application No. 60/523,881 filed Nov. 20, 2003.

FIELD OF THE INVENTION

[0002] The present invention relates to reagents and methods for purifying pertussis toxin (PT).

BACKGROUND OF THE INVENTION

[0003] Pertussis toxin (PT) is produced by Bordetella pertussis is a main component in all vaccines against whooping cough. PT is typically combined with tetanus and diphtheria toxoids. Industrial production of PT is typically achieved by cultivating B. pertussis in defined media. PT is then isolated from the supernatant and purified by using the well-known techniques (i.e., U.S. Pat. Nos. 6,399,076; 5,877,298; and, Sekura, et al. J. Biol. Chem. 258:14647-14651, 1983; Bogdan, et al. Appl. Env. Micro. 69(10): 6272-6279, October 2003). The majority of known methods each require the use of matrix-bound bovine fetuin (BF) or asialofetuin, the source and purity of which is critical. The use of bovine-derived reagents has led to some concern over bovine-related diseases such as bovine spongioform encephalopathy (BSE).

[0004] Those of skill in the art have therefore desired a method for purifying PT that does not rely on BF. One such method is described by Bogdan, et al. (Appl. Env. Micro. 69(10): 6272-6279, October 2003) Peptides having the ability to mimic the glycosidic moiety of bovine fetuin by binding to PT were identified using a phage display system. Three peptides (3G5: NGSFSGF (SEQ ID NO: 1); 3G8: NGSFSGC (SEQ ID NO: 2); and, 3G2: DGSFSGF (SEQ ID NO: 3) having the consensus sequence XGSFSGX (X is any amino acid; SEQ ID NO: 4) were identified as having PT-binding capacity. 3G2 was also utilized in an affinity column to purify PT from a partially purified PT preparation.

[0005] Additional methods for designing and utilizing peptides to purify PT in the absence of bovine products are desired by those of skill in the art. Provided herein are reagents and methodologies for affinity purification of PT without the use of fetuin in any form.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1. A) Schematic representation of the gurmarin library. Positions of the library that are translated to an amino acid sequence are indicated by boxes in light yellow. The sequence of the protein portion (59 amino acids in length) is shown in the single letter amino acid code, where X represents any amino acid. Portions of the library that are not translated are indicated as gray boxes. (a) T7-promoter for optimal in vitro transcription of library, (b) TMV--Tabaco Mosaic Virus translation initiation sequence for perfect in vitro translation of library, (c) His₆-tag for efficient affinity purification of PROfusion® library, (d) structural, flexible linker, (e) gurmarin core with two randomized loops containing 5 and 9 amino acids respectively, (f) structural, flexible linker and (g) optimized linker for efficient coupling with puromycin-acceptor-molecule. B) The construction of the gurmarin PROfusion® library is a multi-step process comprising the following reactions: PCR, in vitro transcription, chemical ligation of RNA with puromycin-oligonucleotide linker, in vitro translation, oligo-dT purification, reverse transcription and His-tag purification.

[0007] FIG. 2. Schematic representation of a PROfusion® selection cycle.

[0008] FIG. 3. Selected gurmarin variants that should be tested for binding activity towards PT. Conserved sequence motifs are highlighted by colored boxes, SEQ ID NOS. 81-101.

[0009] FIG. 4. Sequence analysis of the gurmarin selection round 4 against PT. The amino acid sequence of individual variants is shown in the single letter amino acid code. Constant, flanking regions of the library are indicated by light yellow boxes, constant regions of the gurmarin scaffold are indicated as green boxes. The position of the randomized loops 1 and 2 are indicated. The variants are listed from top to bottom in the following order: SEQ ID NOS. 102(3), 103, 102(3), 104-110, 102, and 111-147.

[0010] FIG. 5. Sequence analysis of the gurmarin selection round 5a against PT (epoxy). The amino acid sequence of individual variants is shown in the single letter amino acid code. Constant, flanking regions of the library are indicated by light yellow boxes, constant regions of the gurmarin scaffold are indicated as green boxes. The position of the randomized loops 1 and 2 are indicated. The variants are listed from top to bottom in the following order: SEQ ID NOS. 148(3), 149, 148(2), 150, 151(2), 148, 155, 148(2), 156, 157, 155, 158(2), 159, 148(4), 160, 148(2), 161, 148(4), 162, 148(2), and 163-169.

[0011] FIG. 6. Sequence analysis of the gurmarin selection round 5b against PT (strep). The amino acid sequence of individual variants is shown in the single letter amino acid code. Constant, flanking regions of the library are indicated by light yellow boxes, constant regions of the gurmarin scaffold are indicated as green boxes. The position of the randomized loops 1 and 2 are indicated. The variants are listed from top to bottom in the following order: SEQ ID NOS. 170, 171(16), 172, 173, 174, 171(2), 175-181, 182(2), 183(2), 184, 185(2), 186(2), 187-195, 196(3), 197, and 198.

[0012] FIG. 7. Sequence analysis of the gurmarin selection round 6a against PT (strep). The amino acid sequence of individual variants is shown in the single letter amino acid code. Constant, flanking regions of the library are indicated by light yellow boxes, constant regions of the gurmarin scaffold are indicated as green boxes. The position of the randomized loops 1 and 2 are indicated. The variants are listed from top to bottom in the following order: SEQ ID NOS. 199 (15), 200, 199 (19), 201, 199 (6), 202, 203(2), 204, 205(2), 206, 207, 203, 208, 209, 203, 210, 211, 204, and 212.

[0013] FIG. 8. Sequence analysis of the gurmarin selection round 6b against PT (strep). The amino acid sequence of individual variants is shown in the single letter amino acid code. Constant, flanking regions of the library are indicated by light yellow boxes, constant regions of the gurmarin scaffold are indicated as green boxes. The position of the randomized loops 1 and 2 are indicated. The variants are listed from top to bottom in the following order: SEQ ID NOS. 213(2), 214-217, 213, 218, 219, 213, 220, 213(2), 221, 213(16), 222, 213(7), 223, 224, 213(2), 225, 213, 226, 213(4), 227-229, 213(5), 229, 213, 231-242, 213, 242, 213, 243, 213, and 244-252.

[0014] FIG. 9. Selected PP26 variants that will be tested for binding activity towards PT. Conserved sequence motifs are highlighted as colored letters. The variants, listed from top to bottom, are SEQ ID NOS. 253-269.

[0015] FIG. 10. Sequence analysis of the PP26 selection round 4 against PT. The amino acid sequence of individual variants is shown in the single letter amino acid code. Constant, flanking regions of the library are indicated by light yellow boxes. The variants are listed from top to bottom in the following order: SEQ ID NOS. 253(6), 270(3), 271, 272, 271(2), 273, 274, 275(2), and 276-305.

[0016] FIG. 11. Sequence analysis of the PP26 selection round 5a against PT (epoxy). The amino acid sequence of individual variants is shown in the single letter amino acid code. Constant, flanking regions of the library are indicated by light yellow boxes. Conserved sequence motifs are highlighted in red or yellow respectively. The variants are listed from top to bottom in the following order: SEQ ID NOS. 306, 307(4), 308-311, 307(3), 312-315, 312 (6), 316, 312, 316, 312 (7), 317-320, 415, 320 (3), 321 (2), 322 (2), 323, 324 (3), 325-338, 339 (2), 341, and 342.

[0017] FIG. 12. Sequence analysis of the PP26 selection round 5b against PT (strep). The amino acid sequence of individual variants is shown in the single letter amino acid code. Constant, flanking regions of the library are indicated by light yellow boxes. Conserved sequence motifs are highlighted in red or yellow respectively. The variants are listed from top to bottom in the following order: SEQ ID NOS. 343 (7), 344-346, 345 (5), 347 (2), 345, 348, 347, 345 (2), 349 (4), 350-354, 355 (2), 356 (2), and 357-367.

[0018] FIG. 13. Sequence analysis of the PP26 selection round 6a against PT. The amino acid sequence of individual variants is shown in the single letter amino acid code. Constant, flanking regions of the library are indicated by light yellow boxes. Conserved sequence motifs are highlighted in red or yellow respectively. The variants are listed from top to bottom in the following order: SEQ ID NOS. 368 (18), 369, 368 (3), 370 (11), 371, 372, 373 (15), 374, 373 (8), 375 (2), 376 (2), 377, 378 (4), 379, 380 (5), and 381-393.

[0019] FIG. 14. Sequence analysis of the PP26 selection round 6b against PT. The amino acid sequence of individual variants is shown in the single letter amino acid code. Constant, flanking regions of the library are indicated by light yellow boxes. The variants are listed from top to bottom in the following order: SEQ ID NOS. 394, 395, 396, 397, (40) 398, 399 (5), 400, 399 (13), 401, 399 (7), 400 (2), 401, 402 (2), 403-414.

[0020] FIG. 15. Immobilization of synthetic biotinylated core peptides to Streptavidin sepharose and verification of binding to purified PT. The unbound fraction of PT was analyzed by separation of 1/40 volume of the supernatant after binding on a 12% NuPage gel with MES running-buffer (upper gel). To analyze sepharose bound PT 50% of the eluate was separated on 12% NuPage gel with MES running-buffer (lower gel). Detection was performed by silver staining Defined amounts of purified PT were used as standard for quantification, except for the gurmarin peptides 15 and 9.

[0021] FIG. 16. Purification of PT out of Sample A (left gel) and Sample B (right gel). To analyze sepharose bound PT 50% of the eluate was separated on 12% NuPage gel with MES running-buffer (lower gel). Detection was performed by silver staining Defined amounts of purified PT were used as standard for quantification, except for the gurmarin peptide 9.

[0022] FIG. 17. Optimization of the washing conditions of bound PT out of sample A or B to immobilized peptides pp26 clone 9 and 15 and gurmarin clone 9 and 15 using 3 washes of 50 mM Tris/HCl, pH 7.5 or 50 mM acetate, pH 6. The PT were analyzed on 12% Bis Tris gels and visualized by silver staining PPM: protein perfect marker.

[0023] FIG. 18. Optimization of washing conditions of bound PT out of sample B to immobilized peptides pp26 clone 9 using 3 to 20 washes of 50 mM Tris/HCl, pH 7.5 or 50 mM acetate, pH 6. The PT was analyzed on 12% Bis Tris gels and visualized by silver staining.

[0024] FIG. 19. Elution of PT from peptide streptavidin sepharose with 0.2 to 2.0 M MgCl₂ in 50 mM Tris/HCl. Peptide bound PT was displaced from the peptide-streptavidin sepharose by three consecutive washes with the indicated elution buffers (20 μl each). Remaining material was subsequently eluted with gel loading buffer. All elutions were analyzed on 12% Bis Tris gels (1×MES running buffer) and visualized by silver staining.

[0025] FIG. 20. Elution of PT from peptide streptavidin sepharose under acidic (50 mM glycine, pH 2.5) or basic (100 mM carbonate buffer, pH 10.5) conditions. Peptide bound PT was displaced from the peptide streptavidin sepharose (20 μl containing ˜200 pmol of one peptide) by three consecutive washes with the indicated elution buffers (40 μl each). Remaining material was subsequently eluted with gel loading buffer. All elutions were analyzed on 12% Bis Tris gels (1×MES running buffer) and visualized by silver staining 1/40 volume of the flow through after peptide streptavidin sepharose incubation with sample A was analyzed was analyzed on the same gel for each peptide.

[0026] FIG. 21. Small scale column purification of PT from sample B on streptavidin sepharose with immobilized pp26 peptide 9 as affinity ligand (A) an gel estimation of the yield of purified PT (B).

[0027] FIG. 22. Small scale column purification of PT from sample B on streptavidin sepharose with immobilized gurmarin peptide 15 as affinity ligand (A) an gel estimation of the yield of purified PT (B).

[0028] FIG. 23. PT binding to peptide streptavidin sepharose in dependence of varying amounts of peptide (as indicated) used for immobilization on streptavidin sepharose (per 1 ml). Amount of bound PT was quantified by direct comparison to defined amounts of purified PT on the same gel. As an example, pp26/9 is plotted against the amount of peptide used for immobilization per ml of streptavidin sepharose. Maximal binding was estimated at approximately 100-150 pmol PT.

[0029] FIG. 24. PT yield as function of varying amounts of input material (sample B) per μl peptide streptavidin sepharose or 6.85 μl asialofetuin sepharose. The amount of eluted PT was calculated on the basis of direct comparison to defined amounts of purified PT on the same gel and listed in the Table 12.

[0030] FIG. 25. Reutilization of peptide sepharose for repeated PT binding and elution. Bound PT to streptavidin sepharose were 4 times eluted with 100 mM Carbonate buffer at pH 10.5 and the column matrix was regenerated with 10 mM HCl.

[0031] FIG. 26. PT elution fractions after FPLC-column purification on pp26/9 peptide streptavidin sepharose (0.5 ml) from sample B. The elution fractions (0.5 μl of each 500 μl elution) were analyzed by PAGE (12% Bis-Tris-Gel, MES running buffer) and silver staining Defined amounts of purified PT were separated on the same gel for direct comparison. Concentration of PT was determined by measuring the absorbance of the elution fractions at 280 nm (A₂₈₀) and compared to purified PT standards (see table).

SUMMARY OF THE INVENTION

[0032] The present invention relates to methods for purifying pertussis toxin (PT). In one embodiment, a method for generating a DNA-protein fusion by covalently bonding a nucleic acid reverse-transcription primer bound to a peptide acceptor to an RNA, translating the RNA to produce a peptide product such that the protein product is covalently bound to the primer, reverse transcribing the RNA to produce a DNA-protein fusion, and testing the fusion product to identify those containing PT binding peptides. The sequence of the peptide is then identified by sequencing. In other embodiments, peptides are provided that have PT-binding capacity and are useful for purifying PT from complex biological fluids. Also provided are peptides bound to solid supports and/or chromatographic media for use in purifying PT from complex biological fluids and methods for carrying out such purifications.

DETAILED DESCRIPTION

[0033] The present invention provides reagents and methodologies for a new method for purifying pertussis toxin (PT). As described above, one such method has been demonstrated by Bogdan, et al. In that method, phage display was utilized to identify PT-binding peptides. For the purposes of practicing the present invention, PT includes naturally expressed PT as well as PT detoxified genetically or otherwise (see, for example, U.S. Pat. Nos. 6,399,076; 6,168,928; 6,018,022; 5,977,304; 5,965,385; 5,856,122; 5,877,298; 5,433,945; 5,358,868; 5,332,583; 5,244,657; 5,221,618; 5,085,862; 4,997,915). In most cases, chemical detoxification is performed following purification of PT. Within this application, all cited references, patents, and patent applications are incorporated herein by reference.

[0034] The present invention also relates to the use of recombinant technology to identify PT-binding peptides. The present invention provides advantages over methods already known in the art. In addition, novel peptides useful in purifying PT are provided herein. In one embodiment, a method for generating a DNA-protein fusion by covalently bonding a nucleic acid reverse-transcription primer bound to a peptide acceptor to an RNA, translating the RNA to produce a peptide product such that the protein product is covalently bound to the primer, reverse transcribing the RNA to produce a DNA-protein fusion, and testing the fusion product to identify those containing PT binding peptides. The sequence of the peptide is then identified by sequencing. In certain embodiments, the RNA moiety may be removed from the complex by treatment with an RNA-degrading compound such as RNase H. Photocrosslinking reagents and peptide acceptors are also useful in practicing the present invention. This system and related reagents have been described elsewhere in, for example, U.S. Pat. No. 6,416,950 (Lohse, et al); U.S. Pat. No. 6,429,300 (Kurz, et al.); U.S. Pat. No. 6,436,665 (Kuimelis, et al.); U.S. Pat. No. 6,602,685 (Lohse, et al); and, U.S. Pat. No. 6,623,926 (Lohse, et al).

[0035] In practicing the invention, a reagent such as a nucleic acid, peptide, fusion, ligand, affinity complex, or the like may be non-diffusively bound or attached to a solid support. In order to be non-diffusively bound or attached, the reagent is chemically or physically combined with the solid support such that the reagent does not move in the presence of liquid from a region of high concentration of reagent to a region of low concentration of reagent. A solid support is any column (i.e., unpacked or packed chromatographic media, column material), bead, test tube, microtiter dish, solid particle (i.e., agarose or sepharose), microchip (i.e., silicon, silicon-glass, or gold chip), membrane (i.e., the membrane of a liposome or vesicle), or other medium to which a reagent may be bound or attached, either directly or indirectly (for example, through other binding partner intermediates such as an antibody, Protein A, Protein G, streptavidin, biotin).

[0036] In preferred embodiments, the reagent is a substance or compound having the ability to bind PT. More preferably, the reagent is a substance or compound having the ability to reversibly bind PT. Even more preferably, the reagent is a peptide having the ability to at least bind, and preferably reversibly bind PT within a liquid containing components other than PT. A reagent that reversibly binds PT is one that binds PT under certain conditions (adsorption), and releases PT under other conditions (desorption). For example, the reagent may bind PT when exposed to conditions of neutral pH and release PT following exposure to conditions of acidic or basic pH. Thus, the ability of the reagent to bind PT (i.e., the equilibrium dissociation constant or K_d) may be manipulated by altering the conditions under which the reagent is in contact with PT. Other conditions may also be changed, including temperature, ionic strength (i.e., salt concentration), solvent concentration, presence or absence of a competitor reagent/free ligand/analogue, polar properties, among others as is known in the art.

[0037] In certain embodiments, an affinity matrix (i.e., a PT-binding peptide bound to a solid support) is utilized to separate a desired component (i.e., PT) from a complex mixture found within a liquid, biological or otherwise. In certain cases, it may be desirable to purify PT from a complex biological fluid such as a bacterial lysate or other composition in which PT does not comprise the majority of components within the fluid (as determined by SDS-PAGE, for example). In other cases, PT may be isolated from a composition that has been partially purified for PT such that the majority of the components within the fluid is represented by PT (a composition consisting of approximately greater than or equal to 50% PT). For example, a composition in which PT consists of about 50% or more of the total protein in the composition as determined by SDS-PAGE would under most circumstances be considered partially purified.

[0038] To purify PT, a composition containing PT may be placed into contact with a PT-binding reagent, preferably a reversibly binding PT-binding reagent, that is bound to a solid support for a sufficient period of time such that PT and the PT-binding reagent bind to one another to form a complex. Non-PT components are then washed away. One or more conditions (i.e., pH) are then changed such that the K_d of the PT-PT binding reagent bond increases, and PT is released from the complex. Released PT is then collected and prepared for further use. Such a separation may be termed affinity purification and products so purified referred to as being affinity purified.

[0039] Chromatographic techniques that are generally considered by those of skill in the art to be less selective than affinity purification techniques may also be used in practicing the present invention. As is known in the art, such techniques may include, for example, size-exclusion chromatography, ion-exchange chromatography, reverse-phase chromatography, and hydrophobic-interaction chromatography. Any of these techniques (including affinity purification) may be carried out using the proper solid support in a low pressure chromotography (LPC), high pressure liquid chromotography (HPLC), or fast protein liquid chromotography (FPLC) setting, for example. Suitable solid supports and equipment for carrying out such techniques are widely available in the art. In practicing the present invention, both affinity chromatography and the more generalized techniques may be combined as needed to either partially purify a starting material (i.e., complex biological fluid such as a bacterial lysate), purify material, or further purify affinity- or otherwise-purified material (i.e., affinity purified PT).

[0040] Peptides have been identified that bind PT and are described herein. Certain peptides have been found to bind PT with high affinity. Such preferred PT binding peptides include:

TABLE-US-00001 (pp26-5; SEQ ID NO: 5) RSSHCRHRNCHTITRGNMRIETPNNIRKDA; (pp26-6; SEQ ID NO: 6) STMNTNRMDIQRLMTNHVKRDSSPGSIDA; (pp26-9; SEQ ID NO: 7) RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA; (pp26-15; SEQ ID NO: 8) RSWRDTRKLHMRHYFPLAIDSYWDHTLRDA; (G-9; SEQ ID NO: 9) SGCVKKDELCARWDLVCCEPLECIYTSELYATCG; (G-10; SEQ ID NO: 10) SGCVKKDELCELAVDECCEPLECFQMGHGFKRCG; (G-15; SEQ ID NO: 11) SGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS; and, (G-19; SEQ ID NO: 12) SGCVKKDELCELAIDECCEPLECTKGDLGFRKCG.

Of these, especially preferred peptides include:

TABLE-US-00002 (pp26-9; SEQ ID NO: 7) RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA; and, (G-15; SEQ ID NO: 11) SGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS.

[0041] Further contemplated are related peptides such as, for example, fragments, variants orthologs, homologues, and derivatives, for example, that possess at least one characteristic or activity (i.e., activity, antigenicity) of the peptide. A fragment comprises a truncation of the sequence (i.e., nucleic acid or polypeptide) at the amino terminus (with or without a leader sequence) and/or the carboxy terminus of the peptide. Fragments may also include variants, orthologs, homologues, and other variants having one or more amino acid additions or substitutions or internal deletions as compared to the parental sequence. In preferred embodiments, truncations and/or deletions comprise about one amino acid, two amino acids, five amino acids, 10 amino acids, 20 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, or more. A variant is a sequence having one or more sequence substitutions, deletions, and/or additions as compared to the parental sequence. Variants may be naturally occurring or artificially constructed. Such variants may be prepared from the corresponding nucleic acid molecules. In preferred embodiments, the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 30, or from 1 to 40, or from 1 to 50, or more than 50 amino acid substitutions, insertions, additions and/or deletions.

[0042] Substitutions may be conservative, or non-conservative, or any combination thereof. Conservative amino acid modifications to the sequence of a polypeptide (and the corresponding modifications to the encoding nucleotides) may produce polypeptides having functional and chemical characteristics similar to those of a parental polypeptide. For example, a "conservative amino acid substitution" may involve a substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position and, in particular, does not result in decreased immunogenicity. Suitable conservative amino acid substitutions are shown in Table I.

TABLE-US-00003 TABLE I Original Preferred Residues Exemplary Substitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleucine Leu Leu Norleucine, Ile, Val, Met, Ala, Phe Ile Lys Arg, 1,4 Diamino-butyric Acid, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Norleucine Leu

[0043] A component such as PT may be said to be purified when it has been separated from at least about 50% of the proteins, lipids, carbohydrates, or other materials with which it is originally found (i.e., a bacterial lysate). It is preferred that the component be separated from at least about 95-100%, 90-95%, 80-90%, 70-80%, 60-70% or 50-60% of the total protein content of a composition. In certain embodiments, a purified component is one that is useful in inducing an immune response in a host to whom the component has been administered, either alone or in combination with other agents. The immune response may include the production of antibodies that bind to at least one epitope of PT or Bordetella pertussis, for example, and/or the generation of a cellular immune response against cells expressing PT. The response may be an enhancement of a current immune response by, for example, causing increased antibody production, production of antibodies with increased affinity for the antigen, or an increased cellular response (i.e., increased T cells). Other measures of an immune response are known in the art and would be suitable in determining whether or not an immune response has occurred.

[0044] PT isolated using the methods described herein may be prepared as pharmaceutical compositions. Preferred pharmaceutical compositions include, for example, PT in a liquid preparations such as a suspensions, syrups, or elixirs. Preferred injectable preparations include, for example, peptides suitable for parental, subcutaneous, intradermal, intramuscular or intravenous administration such as sterile suspensions or emulsions. For example, PT may be prepared as a composition in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The composition may also be provided in lyophilized form for reconstituting, for instance, in isotonic aqueous, saline buffer. Such compositions may also be prepared and utilized as a vaccine as described in, for example, U.S. Pat. Nos. 5,877,298 and 6,399,076 (Vose, et al.) as well as International App. No. PCT/CA96/00278. PT prepared as indicated herein may also be combined with other antigens from disease-causing organisms such as Corynbacterium (i.e., diphtheria), Clostridium (i.e., tetanus), polio virus (i.e., IPV, OPV), hepatitis virus, Neisseria (i.e., meningitis), Streptococcus, Hemophilus, or other pertussis antigens (i.e., filamentous hemagglutinin, pertactin, and agglutinogens), among others as is known in the art.

[0045] A better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration.

EXAMPLES

Materials and Methods

A. Pertussis Toxin (PT)

[0046] PT is a heterooligomeric protein complex with a MWr of 109 kD (consists of the 6 subunits S1, S2, S3, 2×S4, S5). A high purity (>99.99%) preparation, formulated as an ammonium sulfate precipitate, was utilized. A PT-specific ligand (asialofetuin) recognizing the native hexameric complex was also utilized. Asialofetuin is available in a solubilized and in sepharose immobilized form.

B. Gumarin Library Selection

[0047] Gurmarin is a 35-residue polypeptide from the Asclepiad vine Gymnea sylvestre. It has been utilized as a pharmacological tool in the study of sweet-taste transduction because of its ability to selectively inhibit the neural response to sweet tastants in rats. It has no apparent effect in humans. It has been suggested that the taste-suppressing of gurmarin might be due to the peptide either by binding directly to a sweet-taste receptor or interacting with a downstream target in the sweet-taste-transduction system.

[0048] Gurmarin belongs to the family of "knottins", a group of structurally related proteins, typically less than 40 residues in length. Knottins bind to a diverse range of molecular targets that includes proteins, sugars and lipids but share a common scaffold comprising a small triple-stranded antiparallel β-sheet and disulphide bound framework.

[0049] A specialized gurmarin-library was designed with 15 randomized amino acid positions, as shown below:

TABLE-US-00004 Wild-type gumarin: (SEQ ID NO: 13) qqCVKKDELCIPYYLDCCEPLECKKVNWWDHKCig Gumarin core: (SEQ ID NO: 14) CVKKDELCXXXXXXCCEPLECXXXXXXXXXC

Within the gumarin core sequence, X represents any amino acid. This library was validated to yield high affinity binders against protein targets. The gurmarin library combines a set of advantages that makes it the best choice for a selection against the PT-toxin for at least the following reasons: limited flexibility makes up for high entropic cost in conforming to target topology; theoretically fewer amino acids for higher affinities than in linear libraries; resistant to proteases; and susceptibility to redox-elution conditions in downstream applications. The gurmarin library was constructed using process shown in FIG. 1.

1. PCR of Starting Oligonucleotides

[0050] Three gel-purified oligos were used to construct the gurmarin library with two randomized loops. 1 nmole of gurmarin template (≈ca. 610'' sequences) 5'-AGT GGC TCA AGC TCA GGA TCA GGC TGC GTC AAG AAA GAC GAG CTC TGC NNS NNS NNS NNS NNS NNS TGC TGT GAG CCC CTC GAG TGC NNS NNS NNS NNS NNS NNS NNS NNS NNS TGC GGC AGC GGC AGT TCT GGG TCT AGC-3' (SEQ ID NO: 15), was amplified for 6 rounds of PCR (94° C., 1 min; 65° C., 1 min; 72° C., 1 min) using 1 μM of the 5'-His-Tag Primer 5'-TAA TAC GAC TCA CTA TAG GGA CAA TTA CTA TTT ACA ATT ACA ATG CAC CAT CAC CAT CAC CAT AGT GGC TCA AGC TCA GGA TCA-3' (SEQ ID NO: 16) and 1 μM of the 3'-Primer 5'-TTT TAA ATA GCG GAT GCT ACT AGG CTA GAC CCA GAA CTG CCG CT-3' (SEQ ID NO: 17) using Taq-polymerase and analyzed on a 2% agarose gel, which indicated a representative library had been constructed.

2. In Vitro Transcription

[0051] dsDNA was transcribed into RNA using the RiboMax Express In vitro transcription kit from Promega. After incubation for 45 min at 37° C., DNase I was added and the incubation at 37° C. continued for an additional 15 minutes. This mixture was subjected to a phenol/chloroform extraction. Excess of NTPs was removed by NAP-5 gel filtration (Pharmacia). RNA was analyzed on a 6%-TBU-gel, and indicated that the dsDNA had been efficiently transcribed.

3. Chemical Coupling of RNA and Puromycin-Oligonucleotide Linker

[0052] Purified RNA will be annealed (85° C., 1 min cool down to 25° C. at a ramp of 0.3° C./s) to a 1.5-fold excess of puromycin-oligonucleotide linker PEG2A18: 5'-psoralen-UAG CGG AUG C A₁₈ (PEG-9)₂ CC puromycin (SEQ ID NO: 18; nucleotides shown in italics represent 2'-O-methyl-derivatives). The covalent coupling is performed by illumination for 15 min at RT (RT) with UV-light (365 nm). The reaction product was analyzed on 6%-TBU gel and indicated the linking reaction had proceeded efficiently.

4. In Vitro Translation

[0053] Ligated RNA was translated using the rabbit reticulocyte lysate from Promega in the presence of 15 μCi ³⁵S-methionine (1000 Ci/mmole). After a 30 min incubation at 30° C., KCl and MgCl₂ were added to a final concentration of 530 mM and 150 mM respectively and a sample was analyzed on 4-20% Tris/glycine-SDS-PAGE. The gel indicated that the translation reaction was successful.

5. Oligo-dT Purification

[0054] Molecules (mRNA-protein fusions) were isolated by incubation with oligo dT magnetic beads (Miltenyi) in incubation buffer (100 mM Tris-HCl pH 8.0, 10 mM EDTA, 1 mM NaCl and 0.25% Triton X-100) for 5 min at 4° C. PROfusion® molecules were isolated by filtration through MiniMACS-columns (Miltenyi), washing with incubation buffer and elution with water. A sample was analyzed on 4-20% Tris/glycine-SDS-PAGE, and indicated that the reaction was successful.

6. Reverse Transcription

[0055] A corresponding cDNA strand was generated by reverse transcription with SuperScript II Reverse Transcriptase (Gibco BRL) under the manufacture's recommended conditions using a 5-fold excess of 3'-Primer. A sample was analyzed on 4-20% Tris/glycine-SDS-PAGE, and indicated that the reaction was successful.

7. His-Tag Purification

[0056] Reverse transcribed PROfusion® molecules were mixed with Ni-NTA-agarose (50 μl/10 pmole PROfusion®) (QIAGEN) in HBS buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 0.025% Triton X-100, 100 μg/ml sheared salmon sperm DNA, 1 mg/ml BSA) and incubated for 60 min at RT under gentle shaking Ni-NTA was then filtrated, washed with HBS/5 mM imidazole and PROfusions® were eluted with HBS/150 mM imidazole. A sample was analyzed on 4-20% Tris/glycine-SDS-PAGE, and indicated that the purification was successful. 20 pmole (≈ca. 110¹³ sequences) of PROfusion® molecules will be used as input for each selection.

B. Linear Peptide Library PP26 for Selection

[0057] A specialized linear peptide library PP26 with 26 randomized amino acid positions was also designed using the following construct: T7-TMV-MGRGS-HHHHHH-ARS-XXXXXXXXXXXXXXXXXXXXXXXXXX-DANAPK-ASAI (SEQ ID NO: 19). The sequence of the protein portion (50 amino acids in length) is shown in the single letter amino acid code, where X represents any amino acid. Portions of the library that are not translated include: (a) T7: the T7-promoter for optimal in vitro transcription of library; and, (b) TMV: the Tabaco Mosaic Virus translation initiation sequence for perfect in vitro translation of library. MGRGS represents a structural, flexible linker. HHHHHH (SEQ ID NO: 20) represents a His₆-tag for efficient affinity purification of PROfusion® library. ARS represents a second structural, flexible linker. DANAPK (SEQ ID NO: 21) represents a third structural, flexible linker. ASAI represents an optimized linker for efficient coupling with puromycin-acceptor-molecule.

[0058] This library was validated to yield high affinity binders against protein targets. The PP26 library combines two major advantages that makes it an excellent choice for the selection of chromatographic affinity reagents: high flexibility: can conform to the topology of the target; and robustness due to the absence of a conserved structure the resulting binders are resistant to harsh biophysical conditions

1. PCR of Starting Oligonucleotides

[0059] Three gel-purified oligos were used to construct the gurmarin library with two randomized loops. 1 nmole of PP26 template (≈ca. 610¹⁴ sequences) 5'-AGC GGA TGC CTT CGG AGC GTT AGC GTC SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN AGA TCT AGC ATG ATG ATG ATG A-3' (SEQ ID NO: 21), was amplified for 6 rounds of PCR (94° C., 1 min; 65° C., 1 min; 72° C., 1 min) using 1 μM of the 5'-His-Tag Primer 5'-TAA TAC GAC TCA TAG GGA CAA TTA CTA TTT ACA ATT ACA ATG GGA CGT GGC TCA CAT CAT CAT CAT CAT CAT GCT AGA TCT-3' (SEQ ID NO: 22) and 1 μM of the 3'-Primer 5'-AA TTA AAT AGC GGA TGC CTT CGG AGC GTT AGC-3' (SEQ ID NO: 23) using Taq-polymerase and confirmed by analysis on a 2% agarose gel.

2. In Vitro Transcription

[0060] dsDNA was transcribed into RNA using the RiboMax Express In vitro transcription kit from Promega. After incubation for 45 min at 37° C., DNase I was added and the incubation at 37° C. continued for an additional 15 minutes. This mixture was subjected to a phenol/chloroform extraction. Excess of NTPs was removed by NAP-5 gel filtration (Pharmacia). Transcription of RNA was confirmed by analysis on a 6%-TBU-gel.

3. Chemical Coupling of RNA and Puromycin-Oligonucleotide Linker

[0061] Purified RNA will be annealed (85° C., 1 min→cool down to 25° C. at a ramp of 0.3° C./s) to a 1.5-fold excess of puromycin-oligonucleotide linker PEG2A18: 5'-psoralen-UAG CGG AUG C A₁₈ (PEG-9)₂ CC puromycin (nucleotides shown in italics represent 2'-O-methyl-derivatives; SEQ ID NO: 17). The covalent coupling is performed by illumination for 15 min at RT (RT) with UV-light (365 nm). The reaction was confirmed by analysis of the reaction product on 6%-TBU gel.

4. In Vitro Translation

[0062] Ligated RNA was translated using the rabbit reticulocyte lysate from Promega in the presence of 15 μCi ³⁵S-methionine (1000 Ci/mmole). After a 30 min incubation at 30° C., KCl and MgCl₂ were added to a final concentration of 530 mM and 150 mM respectively and translation confirmed by analysis on 4-20% Tris/glycine-SDS-PAGE.

5. Oligo-dT Purification

[0063] Molecules (mRNA-protein fusions) were isolated by incubation with oligo dT magnetic beads (Miltenyi) in incubation buffer (100 mM Tris-HCl pH 8.0, 10 mM EDTA, 1 mM NaCl and 0.25% Triton X-100) for 5 min at 4° C. PROfusion® molecules were isolated by filtration through MiniMACS-columns (Miltenyi), washing with incubation buffer and elution with water. A sample was analyzed to confirm the reaction on 4-20% Tris/glycine-SDS-PAGE.

6. Reverse Transcription

[0064] A corresponding cDNA strand was generated by reverse transcription with SuperScript II Reverse Transcriptase (Gibco BRL) under the manufacture's recommended conditions using a 5-fold excess of 3'-Primer. A sample was analyzed to confirm transcription on 4-20% Tris/glycine-SDS-PAGE.

7. His-Tag Purification

[0065] Reverse transcribed PROfusion® molecules were mixed with Ni-NTA-agarose (50 μl/10 pmole PROfusion®) (QIAGEN) in HBS buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 0.025% Triton X-100, 100 μg/ml sheared salmon sperm DNA, 1 mg/ml BSA) and incubated for 60 min at RT under gentle shaking Ni-NTA was then filtrated, washed with HBS/5 mM imidazole and PROfusions® were eluted with HBS/150 mM imidazole. A sample was analyzed to confirm the reaction on 4-20% Tris/glycine-SDS-PAGE. 20 pmole (≈ca. 110¹³ sequences) of PROfusion® molecules will be used as input for each selection.

C. Target Preparation

[0066] In the PROfusion® technology highly diverse substance libraries, which are composed of up to 10¹³ different PROfusion® molecules (mRNA-Protein fusions), are selected against a wanted target (protein, sugar or lipid) for high affinity binding. In this process the targets will typically be immobilized to solid phases. These solid phase are preferentially magnetic beads that allow fast and efficient handling during the selection process and give low background.

1. Test Targets for Nuclease Activity

[0067] Targets--5 μg PRP and 0.5 μg PT--were contacted with 0.12 pmole radioactive labeled PROfusion® library molecules at 4° C. and RT (RT) followed by an incubation for 1 h and 16 h respectively. The integrity of PROfusion® molecules after incubation was confirmed by 4-20% Tris/glycine SDS-PAGE and subsequent autoradiography. Degradation of PROfusion® molecules was not detected, thus demonstrating that the targets are free of nucleases.

2. Test Targets for Protease Activity

[0068] Targets--5 μg PRP and 0.5 μg PT--were contacted with 1 μg purified GST-protein at 4° C. and RT followed by an incubation for 1 h and 16 h respectively. The integrity of GST-protein after incubation was analyzed by 4-20% Tris/glycine SDS-PAGE and subsequent Coomassie Brilliant Blue staining Degradation of GST-protein was not detected, thus demonstrating that the targets are free of proteases.

D. Immobilization of PT

1. Reconstitution of PT

[0069] 500 μl of the precipitate (2.26 mg/ml) as delivered by Aventis Pasteur were centrifuged at 21.400×g for 45 min at RT. The supernatant was discarded; the pellet was dissolved in 1100 μl CTW-buffer (0.286 g NaHCO₃, 0.170 g Na₂CO₃, 50 μl Tween-80, add to 50 ml MilliQ H₂O). To check the quality of this PT preparation a dilution series (250 ng, 500 ng, 1 μg, 2.5 μg, 5 μg and 15 μg) was separated on a 4-12% BisTris SDS-PAGE, run in MES-buffer). At least 4 bands could be clearly separated, corresponding to the subunits S1 (28 kD), S2 (23 kD), S3 (22 kD) and S4 (11.7 kD). The smallest protein S5 (9.3 kD) in the PT-complex could not be seen. Probably, this band co-migrates in this gel system with the only slightly larger S4 subunit.

2. Coupling Strategy

[0070] Several methods were established for immobilization of proteins to magnetic particles. In principle two major strategies are used: primary amino groups and sulfhydryl groups of the target protein are tethered covalently to epoxy-activated magnetic beads (Dynal) forming stabile amide or thioether bounds. This reaction is performed in the presence of ammonium sulfate to promote the reaction and typically results in a very efficient coupling of the target protein. Anyhow, certain proteins seem to undergo structural changes under these conditions resulting in a bound but not native and/or inactive conformation; and, primary amino groups and sulfhydryl groups of the target protein are tethered covalently to NHS-ester activated biotin derivatives (Pierce) subsequently followed by an immobilization of now biotinylated protein to streptavidin magnetic beads (Dynal)

[0071] Typically, covalent coupling of a target protein to epoxy beads is preferred if reaction conditions are suitable for a given target since this method guarantees that only the target is presented on the beads. In the case of a biotin/streptavidin coupling the beads also present streptavidin that could lead to the enrichment of anti-streptavidin binder during a selection. Therefore, Phylos has established specialized methods to preclear PROfusion® libraries for streptavidin binders to get high quality results for a given target. But in total a covalent coupling typically results in a faster enrichment of target specific binders. In the specific case of PT it is most reasonable to start with a covalent coupling strategy since it is known that ammonium sulfate incubation does not influence the functionality of the PT-protein.

3. Optimization of Coupling Conditions to Epoxy Beads (Dynal)

[0072] The coupling conditions for PT were optimized in several independent experiments (different ammonium sulfate concentrations (0.5-2.0 M) and different beads/target-ratios were applied, as well as time- and temperature dependency (2 min-16 h; 8° C.-RT). Best results were observed for the following reaction condition: A final volume of 300 μl, consisting of 100 μg PT, 3.310⁸ beads and a final ammonium sulfate concentration of 1M was incubated in a time course for 2 min to 60 min at RT in a 2 ml Eppendorf tube. After incubation the tube was placed in a magnet for 4 min to pull down the beads and the supernatant was stored for subsequent gel analysis. The beads were washed once with 1 ml HEPES-buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 0.025% Triton X100) and an aliquot of beads (5% of the beads) were analyzed on a 4-12% BisTris SDS-PAGE to determine the amount of associated protein. It was found that coupling of PT to epoxy beads occurs very efficiently even after only a two minute reaction.

4. Semi-Preparative Coupling of PT to Epoxy Beads

[0073] 2.6 mg dry epoxy-activated beads (M-270, Dynal) (˜1.710⁸ beads) were resuspended in 1 ml phosphate buffer (19 mM NaH₂PO₄, 81 mM Na₂HPO₄, pH 7.4) and equilibrated for 10 min. The equilibration was repeated two times with fresh phosphate buffer. Subsequently the beads were directly used in a coupling reaction with 480 pmole reconstituted PT (1 μg/μl in CTW buffer) in 1 M ammonium sulfate (final volume 157 μl). After incubation at RT for 15 min under continuos agitation the beads were washed with 300 μl HBS-buffer, followed by three washing steps with HEPES-buffer and finally resuspended in 240 μl HEPES-buffer and stored in aliquots at 4° C. The effectiveness of the coupling reaction was checked by a SDS-polyacrylamidgel-analysis of all wash fractions, the supernatant of the coupling reaction and the fraction of PT which was removable from the washed beads by SDS-loading-buffer.

5. Analysis of Epoxy-Bead Immobilized PT for its Binding to Asialofetuin

[0074] 40 μl of the PT-derivatized beads were incubated with 320 pmole asialofetuin in HEPES-buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 0.025% Triton-X100) for 1 h at RT (final reaction volume 200 μl), washed 2-7 times with 200 μl HEPES-buffer and finally resuspended in 30 μl HEPES-buffer. 50% of the beads were analyzed on SDS-PAGE to confirm the reaction.

[0075] Tests of these PT-derivatized beads after one week of storage at 4° C. showed a reduced asialofetuin binding capacity indicating that the material looses its performance by long term storage. Thus, PT-derivatized beads have to be prepared fresh and quality controlled for each selection round. Since this procedure is quite time consuming, an alternative immobilization strategy involving a biotinylation of PT was evaluated.

6. Semi-Preparative Biotinylation of PT

[0076] A biotinylation reaction was performed by incubation of 0.4 mg (˜3.65 nmole) reconstituted PT (1 μg/μl in CTW buffer) with 25 μg EZ-link-sulfo-NHS-LC-LC-biotin (PIERCE) in a final volume of 740 μl 50 mM HEPES, 150 mM NaCl, 0.2% Triton-X100. After an incubation period of 2 h on ice under permanent agitation the biotinylation reaction was quenched by addition of 74 μl 1M Tris/HCl pH 7.0. Subsequently, the protein was dialyzed against HEPES-buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 0.025% Triton X100) at 4° C. using a Slide-a-lyzer cassette (PIERCE, 3500 MWCO 0.5-3 ml) to remove the excess of biotinylation reagent. The biotinylated PT was removed from the dialysis cassette and stored in aliquots at 20° C.

7. Quality Control of Biotinylated PT Using a BIAcore Instrument

[0077] The quality of the biotinylation reaction was controlled by analysis of the interaction of biotinylated PT with a BIAcore streptavidin chip using BIAcore instrument (BIAcore 2000). It was also possible to detect the binding of asialofetuin to chip immobilized biotinylated PT (binding signal of ˜400 RU to immobilized PT; unspecific binding of ˜100 RU to the control cell).

F. Analysis of Biotinylated PT for Binding to Streptavidin Magnetic Beads and to Asialofetuin

1. Binding of Biotinylated PT to Streptavidin Magnetic Beads

[0078] 20 μl streptavidin magnetic beads (Dynal) were incubated with 20 pmole of biotinylated PT in 1×HBS-buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 1 mg/ml BSA, 100 μg/ml salmon sperm DNA, 0.025% Triton-X100) for 1 h at RT, washed 3× with HEPES-buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 0.025% Triton X100) and resuspended in 16 μl SDS-gel-loading buffer. 8 μl were analyzed by SDS-PAGE to confirm conjugation. In a negative control experiment under comparable conditions, free PT (not biotinylated) did not interact with streptavidin magnetic beads.

2. Binding of Asialofetuin to Bead Immobilized Biotinylated PT

[0079] 20 μl streptavidin magnetic beads (Dynal) were incubated with 20 pmole of biotinylated PT in 1×HBS-buffer for 1 h at RT, washed 4× with HEPES-buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 0.025% Triton X100). Subsequently, beads with immobilized biotinylated PT were incubated with 40 pmole asialofetuin in HEPES-buffer for 1 h at RT. After 4 washes with HEPES-buffer beads were resuspended in 16 μl SDS-gel-loading buffer. 8 μl were analyzed by SDS-PAGE to confirm binding. A simultaneous incubation of biotinylated PT and asialofetuin to the streptavidin magnetic beads instead of serial incubations resulted as well in binding of asialofetuin to biotinylated PT. In a comparable control experiment, it was determined that asialofetuin did not interact with the streptavidin magnetic beads non-specifically. Similar quality controls with biotinylated PT that has been stored for one week at 20° C. showed no significant decrease in streptavidin and/or asialofetuin binding competence. Therefore, biotinylated PT was used as standard target in subsequent selections.

Example 2

Isolation of Peptides Selective for PT or PRP

[0080] The gurmarin PROfusion® library and the targets PT and PRP immobilized to magnetic beads were then contacted under strictly controlled stringency conditions. These conditions allow predominately those variants of the PROfusion® library showing elevated affinity for PT or PRP, respectively, to bind to the targets. After extensive washes that dilute unwanted, non-specific binding variants, the bound PROfusion®-molecules are eluted from the beads and are subjected to a new PROfusion®-formation cycle as shown in (FIG. 2). By successive rounds of selection and re-amplification along with a fine adaptation of stringency conditions a population of highly specific binding molecules to the given target is enriched (10). Subsequently the DNA-portion of this population is cloned into an E. coli plasmid vector to isolate individual variants that can be analyzed in detail by sequencing.

[0081] Six successive selection rounds against immobilized PT have been performed with the gurmarin PROfusion®-library. According to the perception described above, biotinylated PT immobilized to streptavidin beads has been used in these selections (Table 1). In selection round 4, a low background binding of the gurmarin pool to streptavidin beads has been observed which might indicate a starting enrichment of bead and/or streptavidin binding gurmarin variants. Therefore, in the following fifth selection round two individual selections were performed using biotin/streptavidin immobilized PT as target and epoxy bead coupled PT, respectively. In both selections, a clear background corrected enrichment of target binding was observed (Table 1). This trend has been confirmed in the sixth selection round using biotin/streptavidin immobilized PT, clearly indicating an accumulation of PT-binding variants (Table 1).

A. Cloning of Selected Gurmarin Binder Pools

[0082] The gurmarin DNA-pools resulting from selection rounds R4, R5 and R6 were cloned into the pCR®2.1-TOPO®-vector using the TOPO TA Cloning® kit (Invitrogen). The gurmarin DNA was ligated to the pCR®2.1-TOPO®-vector in different concentrations. For 6 μl reactions, 0.5 μl, 2 μl and 4 μl of the gurmarin pool DNA were used respectively. The ligation was performed according to the manufacturer's instructions.

[0083] Two (2) μl of these ligations were transformed into 20 μl of the E. coli Top 10 F' competent cells (Invitrogen) and spread out on LB plates containing 50 μg/ml Kanamycin and 0.5% Glucose. From each of these transformations 150 single colonies were picked to a masterplate containing 50 μg/ml Kanamycin and 0.5% Glucose to repress T7 dependent protein expression and a second plate containing X-Gal and IPTG for a blue white screening. For each transformation, 96 of the colonies from the repressed masterplate corresponding to the white colonies from the blue white test were used to inoculate a 96 well LB agar plate and 500 μl liquid cultures (LB containing 50 μg/ml Kanamycin and 0.5% Glucose). The 96 well agar plates were sent out for commercial sequencing service. The liquid cultures were mixed with 500 μl 40% Glycerol, frozen in liquid nitrogen and stored at -80° C.

[0084] From each individual clone, plasmid DNA was prepared and subjected to an automated DNA-sequencing procedure using a M13-primer 5'-TGT AAA ACG ACG GCC AGT-3' (SEQ ID NO: 24). As shown in FIGS. 3-8, a single gurmarin sequence variant begins to be significantly enriched in selection round 4 and represents >90% of all sequences after selection round 6. This clearly indicates that this variant probably binds with the highest affinity to PT. In addition to this most prominent sequence variant, a variety of other gurmarin sequences have been enriched that partially share common sequence motifs. This finding indicates that these other sequences show affinity towards PT as well.

B. PP26 Affinity Selection Against Immobilized PT

[0085] In parallel to the gurmarin selection six successive selection rounds against immobilized PT have been performed with the PP26 PROfusion®-library. Biotinylated PT immobilized to streptavidin beads has been used in these selections (Table 2). In selection round 4, a low background binding of the gurmarin pool to streptavidin beads has been observed which might indicate a starting enrichment of bead and/or streptavidin binding PP26 variants. Therefore, in the following fifth selection round two individual selections were performed using on the one hand biotin/streptavidin immobilized PT as target and on the other hand epoxy bead coupled PT. In both selections a clear background corrected enrichment of target binding have been detected (Table 2). This trend was confirmed in the sixth selection round using biotin/streptavidin immobilized PT, thus, clearly indicating an accumulation of PT-binding variants.

C. Cloning of Selected PP26 Binder Pools

[0086] The PP26 DNA-pools resulting from selection rounds R4, R5 and R6 were cloned into the pCR®2.1-TOPO®-vector using the TOPO TA Cloning® kit (Invitrogen). The PP26 DNA was ligated to the pCR®2.1-TOPO®-vector in different concentrations. For 6 μl reactions 0.5 μl/2 μl and 4 μl of the gurmarin pool DNA were used respectively. The ligation was performed according to the manufacturer's instructions. 2 μl of these ligations were transformed into 20 μl of the E. coli Top 10 F' competent cells (Invitrogen) and spread out on LB plates containing 50 μg/ml Kanamycin and 0.5% Glucose. From each of these transformations 150 single colonies were picked to a masterplate containing 50 μg/ml Kanamycin and 0.5% Glucose to repress T7 dependant protein expression and a second plate containing X-Gal and IPTG for a blue white screening. For each Transformation 96 of the colonies from the repressed masterplate corresponding to the white colonies from the blue white test were used to inoculate a 96 well LB agar plate and 500 μl liquid cultures (LB containing 50 μg/ml Kanamycin and 0.5% Glucose). The 96 well agar plates were sent out for commercial sequencing service. The liquid cultures were mixed with 500 μl 40% Glycerol, frozen in liquid nitrogen and stored at -80° C.

D. Sequencing of Individual Binder Variants

[0087] From each individual clone plasmid DNA was prepared and subjected to an automated DNA-sequencing procedure using a M13-primer 5'-TGT AAA ACG ACG GCC AGT-3' (SEQ ID NO: 24). As shown in FIGS. 9-14, two main variants have been enriched during the selection rounds. Both variants share a common conserved sequence motif. This finding indicates that the side chains of the conserved amino acids putatively establish a direct interaction with a certain PT surface region. Furthermore, at least 4 additional variants have been enriched at lesser extent. Since these variants do not comprise the above mentioned conserved sequence motif it can be concluded that these variants potentially bind to different surface regions of PT.

E. Validation of Selected PT-Binding Gurmarin- and PP26-Variants

[0088] Since the selections were performed with PROfusion®-molecules--mRNA-peptide-fusions--it is necessary in the first step of the post selection analysis to check the free peptides for their ability to bind do the target. In the next step, those variants that establish their target binding through the peptide and not the nucleic acid portion are subjected to a specificity test in the presence of AP process fluids. By this measure, those variants should be identified that are most suitable to the AP process.

1. Test of Free Peptides for their Binding Capacity to PT

[0089] For a qualitative binding assay of free peptides of single enriched gurmarin- and PP26-binder variants the TNT T7 coupled Reticolocyte Lysat System (Promega #L5540) was used, as follows. DNA of single binder candidates was amplified by colony-PCR out of the glycerol stock of binder clones. To avoid mutations during PCR a proofreading polymerase (Pwo) was used. The PCR products were analyzed on a 2% agarose gel. 5.0 μl of PCR product were used as template for coupled in vitro transcription/translation reaction using the TNT system in a final volume of 53 μl according to the manufacturers instructions. Expressed binder candidates were subsequently purified by Ni-NTA chelat chromatography (QIAGEN). Radioactively labeled His-tag purified binder candidates (˜40-70 fmol of each peptide) were incubated with biotinylated PT immobilized on streptavidin-magnetic beads for 1 h at RT. The beads were washed 3× with HBS-buffer and then resuspended in water and analyzed by liquid scintillation counting. In control experiments each candidate was incubated with streptavidin beads only (without PT). The best binder candidates of PP26 and gurmarin were identified (Tables 3 and 4, below) and were subjected to the following specificity test.

2. Specificity Test of Gurmarin and PP26 Variants in the Presence of Process Fluids

[0090] For a semi-quantitative binding and specificity assay of free gurmarin and PP26 peptides in the presence of Aventis Pasteur process fluids the peptides were first produced as PROfusion®, purified to homogeneity and than transferred to free peptides by an S1-nuclease digest. For amplification of a sufficient amount of DNA of the selected binder variants (10 Gurmarin clones and 7 PP26 clones) a PCR was performed using a PCR product from TNT expression as template. After 10 cycles of PCR (94° C., 30 sec; 60° C., 30 sec; 72° C., 30 sec) the samples were analyzed on a 2% agarose gel. dsDNA (PCR product) was transcribed into RNA using the RiboMax Express In vitro transcription kit from Promega. After incubation for 45 min at 37° C., DNase I was added and the incubation at 37° C. continued for an additional 15 minutes. This mixture was subjected to a phenol/chloroform extraction. Excess of NTPs was removed by NAP-5 gel filtration (Pharmacia). RNA was analyzed on a 6%-TBU-gel.

[0091] Purified RNA was annealed (85° C., 1 min cool down to 25° C. at a ramp of 0.3° C./s) to a 1.5-fold excess of puromycin-oligonucleotide linker PEG2A18: 5'-psoralen-UAG CGG AUG C A₁₈ (PEG-9)₂ CC puromycin (nucleotides shown in italics represent 2'-O-methyl-derivatives; SEQ ID NO: 17). The covalent coupling was performed by illumination for 15 min at RT (RT) with UV-light (365 nm). The reaction product was analyzed on 6%-TBU gel. Ligated RNA was translated using the rabbit reticulocyte lysate from Promega in the presence of 15 μCi ³⁵S-methionine (1000 Ci/mmole). After a 30 min incubation at 30° C., KCl and MgCl₂ were added to a final concentration of 530-mM and 150 mM respectively and a sample was analyzed on 4-20% Tris/glycine-SDS-PAGE. mRNA-protein fusions (PROfusions®) were isolated by incubation with oligo dT magnetic beads (Miltenyi) in incubation buffer (100 mM Tris-HCl pH 8.0, 10 mM EDTA, 1 mM NaCl and 0.25% Triton X-100) for 5 min at 4° C. PROfusion® molecules were isolated by filtration through MiniMACS-columns (Miltenyi), washing with incubation buffer and elution with water. A sample was analyzed on 4-20% Tris/glycine-SDS-PAGE.

[0092] To remove the mRNA part of the mRNA-protein fusions the oligo dT purified molecules were digested with S1-Nuclease (S1-Nuclease cleaves the DNA-part of the Puromycin linker) according to the manufacturers instructions. Samples of the PROfusion molecules before and after S1-digest were analyzed on 4-12% Bis/Tris SDS-PAGE. Streptavidin beads (M280 Dynal) were washed in HBS and incubated o/n at 4° C. Biotinylated PT (900 pmol) was incubated with 900 μl Strepbeads (preblocked in HBS buffer) for 1 h at RT. After immobilization of PT, the beads were blocked with biotin (2 mM biotin in HBS) for 1 min and immediately washed 4× with HBS buffer to remove any traces of biotin. Control beads (without PT) were blocked with biotin in the same way.

[0093] For binding analysis of the selected peptides several parallel reactions were set up, as follows: negative control only with biotin blocked Streptavidin beads; positive control with PT immobilized on Streptavidin beads; background control with biotin blocked beads in combination with 1/4 volume Aventis Pasteur sample-solution C (flow through 1. AF column); mix of PT in combination with 1/4 volume of sample-solution C; background control with biotin blocked beads in combination with 1/4 volume of Aventis Pasteur sample-solution E (culture medium); mix of PT in combination with 1/4 volume of sample-solution E; reactions 3-6 were performed to investigate the capacity of the selected peptides to bind PT specifically in the presence of samples provided by Aventis Pasteur. Binding was done for 1 h at RT in the presence of a protease inhibitor mix (complete Mini® ROCHE), to avoid degradation of the peptides. After washing with HBS solution the beads were analyzed by scintillation counting.

[0094] As shown in Table 3, three (#9, 10 19) of the ten tested gurmarin variants show a target binding to PT that is not influenced by any of the AP process fluids. These variants are the most promising candidates for affinity chromatographic applications within the AP process.

[0095] As shown in Table 4 three (#5, 6 9) of the seven tested PP26 variants show target binding to PT that is not reduced by the AP process fluids. These variants are the most promising candidates for further affinity chromatographic applications within the AP process.

TABLE-US-00005 TABLE 3 Post selection analysis of gurmarin-variants* # seq # peptide sequence test 1 test 2 SEQ ID 1 194227 MHHHHHHSGSSSGSGCVKKDELCAGSVGHCCEPLECLRRFLNLRWCGSGSSGSS -- n.d. 26 2 194238 MHHHHHHSGSSSGSGCVKKDELCIVMRAPCCEPLECLRRYMLKHMCGSGSSGSS -- n.d. 27 3 194239 MHHHHHHSGSSSGSGCVKKDELCKAFRYSCCEPLECLRKWLKARFCGSGSSGSS -- n.d. 28 4 194251 MHHHHHHSGSSSGSGCVKKDELCLRSSIDCCEPLECLYKWMQRRLCGSGSSGSS -- n.d. 29 5 194210 MHHHHHHSGSSSGSGCVKKDELCWPRRHKCCEPLECLLEMLERKRCGSGSSGSS -- n.d. 30 6 194261 MHHHHHHSGSSSGSGCVKKDELCMSMACVCCEPLECKYHGYFWLCGSGSSGSS -- n.d. 31 7 194214 MHHHHHHSGSSSGSGCVKKDELCAVWFDVCCEPLECTYQSGYYWLCGSGSSGSS -- n.d. 32 8 194226 MHHHHHHSGSSSGSGCVKKDELCEPWYWRCCEPLECVYTSGYYYSCGSGSSGSS -- n.d. 33 9 194259 MHHHHHHSGSSSGSGCVKKDELCARWDLVCCEPLECIYTSELYATCGSGSSGSS 34 12 194297 MHHHHHHSGSSSGSGCVKKDELCVFYFPNCCEPLECRWVNDNYGWCGSGSSGSS -- 35 13 194330 MHHHHHHSGSSSGSGCVKKDELCMSMACVCCEPLECKYHGYFWLCGSGSSGSS -- 36 14 194479 MHHHHHHSGSSSGSGCVKKDELCTTASKSCCEPLECKWTNEHFGTCGSGSSGSS -- 37 15 194511 MHHHHHHSGSSSGSGCVKKDELCSQSVPMCCEPLECKWFNENYGICGSGSSGSS -- 38 16 194533 MHHHHHHSGSSSGSGCVKKDELCARWDLVCCEPLECIYTSELYATCGSGSSGSS -- 39 17 194486 MHHHHHHSGSSSGSGCVKKDELCARWDLVCCEPLECLGHGLGYAYCGSGSSGSS n.d. 40 18 194668 MHHHHHHSGSSSGSGCVKKDELCMWSREVCCEPLECYYTGWYWACGSGSSGSS -- 41 10 194264 MHHHHHHSGSSSGSGCVKKDELCELAVDECCEPLECFQMGHGFKRCGSGSSGSS 42 19 194737 MHHHHHHSGSSSGSGCVKKDELCELAVDECCEPLECTKGDLGFRKCGSGSSGSS 43 20 194716 MHHHHHHSGSSSGSGCVKKDELCELAIDVCCEPLECLGHGLGYAYCGSGSSGSS n.d. 44 21 194720 MHHHHHHSGSSSGSGCVKKDELCELAIDVCCEPLECLGHGLGYAYCGSGSSGSS -- -- 45 11 194328 MHHHHHHSGSSSGSGCVKKDELCNWVTPMRCEPLECLGHGLGYAYCGSGSSGSS n.d. 46 *Test 1 represents the target binding ability of free peptides (0) and test 2 represents the binding specificity of variants in the presence of AP process fluids (0). Variants that are positive in both assays are 9, 10, and 19.

TABLE-US-00006 TABLE 4 Post selection analysis of PP26-variants* # seq # peptide sequence test 1 test 2 SEQ ID 1 197569 MGRGSHHHHHHARSDWELSPPHVAITTRHLINCTDGPLLRDANAPKASAI -- n.d. 47 2 197536 MGRGSHHHHHHARSLNGESTSNILTTSRKVTEWTGYTASVDANAPKASAI -- n.d. 48 3 197611 MGRGSHHHHHHARSQVTWHHLADTVTTKNRKCTDSYIGWNXANAPKASAI -- n.d. 49 4 197530 MGRGSHHHHHHARSIIVIHNAIQTHTPHQVSIWCPPKHNRDANAPKASAI -- n.d. 50 5 197557 MGRGSHHHHHHARSSHCRHRNCHTITRGNMRIETPNNIRKDANAPKASAI 51 6 197596 MGRGSHHHHHHARSTMNTNRMDIQRLMTNHVKRDSSPGSIDANAPKASAI 52 7 197552 MGRGSHHHHHHARSLSALRRTERTWNTIHQGHHLEWYPPADANAPKASAI -- n.d. 53 8 197541 MGRGSHHHHHHARSWTSMQGETLWRTDRLATTKTSMSHPPDANAPKASAI -- n.d. 54 9 197588 MGRGSHHHHHHAPSNVIPLNEVWYDTGWDRPHRSRLSIDDDANAPKASAI 55 10 197635 MGRGSHHHHHHARSCLATRNGFVMNTDRGTYVKRPTVLQDANAPKASAI -- 56 11 197797 MGRGSHHHHHHARSWGLSGTQTWKITKLATRLHHPEFETNDANAPKASAI -- n.d. 57 12 197888 MGRGSHHHHHHARSWRWHNWGLSDTVASHPDASNSLNMMYDANAPKASAN -- n.d. 58 13 197897 MGRGSHHHHHHLDLWGPPSGSPRTRSTTGTSTTSSPSTPGTLTLRRHPH -- n.d. 59 14 197825 MGRGSHHHHHHARSWQPEVKMSSLVDTSQTVGAAVETRTTDANAPKASA -- 60 15 198000 MGRGSHHHHHHARSWRDTRKLHMRHYFPLAIDSYWDHTLRDANAPKASAI -- 61 16 197983 MGRGSHHHHHHARSWTSMQGETLWRTDRLATTKTSMSHPPDANAPKASAI -- n.d. 62 17 197998 MGRGSHHHHHHHARSPLWYHYNCWDTICLADWLKDRPHGVYDANAPKASA -- n.d. 63 18 197947 MGRGSHHHHHHARSVGTTIRIAQDTEHYRNVYHKLSQYSRDANAPKASAI -- 64 19 197954 MGRGSHHHHHHARSVGTTIRIAQDTEHYRNVYHKLSQYSRDANAPKASAI -- n.d. 65 20 197971 MGRGSHHHHHHARSNVIPLNEVWYDTGWDRPHRSRLSIDDDANAPKASAI -- n.d. 66 *Test 1 represents the target binding ability of free peptides (0) and test 2 represents the binding specificity of variants in the presence of AP process fluids (0). Variants that are positive in both assays are 5, 6 and 9.

F. Peptide Production by Chemical Synthesis

[0096] Eight different peptides were produced by chemical synthesis in form of N-terminal biotinylated peptides. The Biotin group was spaced via a short hydrophilic linker (PEG2=8-Amino-3,6-dioxaoctanoic acid). Two of these 8 peptides (PP26-5c and gumarin-9c) were additional synthesized in form of C-terminal tagged biotinylated peptides (via an additional C-terminal Lysine). The peptides were automatically synthesized using the Fmoc/But strategy according to Sheppard, purified by HPLC and subsequently lyophilized. The quality of all purified peptides was confirmed by mass spectroscopy. The target quantity of each peptide synthesis was 5 mg purified peptide. An overview about yield and purity of the synthetic peptides after purification is given in Table 5.

TABLE-US-00007 TABLE 5 PeptideSynthesis of Pertussis Toxin Binding Peptides* Purity Yield Selection Clone Seq # Sequence (%) (mg) SEQ ID pp26 5 c 197557 RSSHCRHRNCHTITRGNMRIETPNNIRKDAK 90-95 7, 7 67 pp26 5 n 197557 RSSHCRHRNCHTITRGNMRIETPNNIRKDA 90-95 7, 6 68 pp26 6 n 197596 RSTMNTNRMDIQRLMTNHVKRDSSPGSIDA 90-95 6, 3 69 pp26 9 n 197588 RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA 90-95 5, 8 70 pp26 15n 198000 RSWRDTRKLHMRHYFPLAIDSYWDHTLRDA 90-95 4, 8 71 gurmarin 9 c 194259 SGCVKKDELCARWDLVCCEPLECIYTSELYATCGK 70 1, 0 72 gurmarin 9 n 194259 SGCVKKDELCARWDLVCCEPLECIYTSELYATCG 80-90 4, 0 73 gurmarin 10 n 194264 SGCVKKDELCELAVDECCEPLECFQMGHGFKRCG 90-95 4, 9 74 gumarin 15n 194511 SGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS 90-95 6, 3 75 gurmarin 19 n 194737 SGCVKKDELCELAIDECCEPLECTKGDLGFRKCG 90-95 6, 7 76 *Abbreviation c in the clone name indicates C-terminal biotinylated peptides, abbreviation n indicates N-terminal biotinylated peptides.

[0097] All pp26 peptides were dissolved in 100 mM HEPES, pH 7.4, 200 mM NaCl with a final concentration of 100 μM. All gurmarin peptides were dissolved in 100 mM HEPES, pH 7.4, 200 mM NaCl, 2 mM GSH, 1 mM GSSG with a final concentration of 100 μM and subsequently incubated under nitrogen for at least 48 hours to allow structural folding.

G. Peptide Production by Bacterial Expression

[0098] The peptides which were identified as binders to the pertussis toxin were subcloned in frame to glutathione-S-transferase (GST) and expressed bacterially. The GST-tag enhances the solubility and allows purification using Glutathione Sepharose. An engineered protease cleavage site recognized by the specific PreScission® protease allows removal of the GST-tag releasing the peptide. The PreScission® protease itself is a fusion protein of GST and human rhinovirus (HRV) type 14 3C protease and specifically recognizes the sequence Leu-Phe-Gln*Gly-Pro cleaving between the Gln and Gly residues. After the cleavage the uncleaved product as well as the protease can be removed from the cleavage reactions using Glutathione Sepharose.

H. Construction of Expression Vectors

[0099] 1. Construction of GST Fusions for pp26-Variants

[0100] As template for PCR served the pCR2.1 vector containing the sequences of the identified pp26 binders to PT. The products obtained in a PCR using the oligonucleotides #467 (5'-CATGCCATGGGACGTGGCTCACATCATC-3'; SEQ ID NO: 66) and #468 (5''-phosphate-GGGTTAAATAGCGGATGCCTTCGGAGCGTTAGCGTC-3''; SEQ ID NO: 67) with Pwo DNA polymerase (Roche) were digested with NcoI (New England Biolobs). A modified vector (pGEX6P (Amersham/Pharmacia) containing an additional NcoI site) was digested with NcoI/SmaI (New England Biolobs) and the PCR product was directionally cloned into the NcoI/SmaI site of this vector. After transformation in TOP10 (Invitrogen) positive clones were identified by colony PCR and verified by sequencing.

2. Construction of GST Fusions for Gurmarin-Variants

[0101] As template for PCR served the pCR2.1 vector containing the sequences of the identified gurmarin binders to PT. The products obtained in a PCR using the oligonucleotides #464 (5''-GGAGATCTCATATGCACCATCACCATCACCATAGTGGC-3''; SEQ ID NO: 68) and #465 (5'-phosphate-GGGTTAAATAGCGGATGCTACTAGGC-3'; SEQ ID NO: 69) with Pwo DNA polymerase (Roche) were digested with NdeI (New England Biolobs). A modified vector (pGEX6P (Amersham/Pharmacia) containing an additional NdeI site) was digested with NdeI/SmaI (New England Biolobs) and the PCR product was directionally ligated into the Nde/SmaI site of this vector. After transformation in TOP10 (Invitrogen) positive clones were identified by colony PCR and verified by sequencing (Table 6).

TABLE-US-00008 TABLE 6 Vectors used for bacterial expression Plasmid number pp26 pS840 pGEX6P-(His)₆-pp26 K5 pS850 pGEX6P-(His)₆-pp26K6 pS841 pGEX6P-(His)₆-pp26K9 pS842 pGEX6P-(His)₆-pp26K15 gurmarin pS836 pGEX6P-(His)₆-gurmarin K9 pS837 pGEX6P-(His)₆-gurmarin K10 pS838 pGEX6P-(His)₆-gurmarin K15 pS839 pGEX6P-(His)₆-gurmarin K19

3. Expression and Purification of GST-pp26 Fusions

[0102] The bacterial strain Rosetta (DE3) pLysS (Novagen) was transformed with plasmid DNA (see Table). The transfomands of the pp26 variants were grown at 37° C. 250 rpm to an OD₆₀₀ of ˜0.5 and induced by the addition of 1 mM IPTG for 4 h. In case of gurmarin-GST-fusions the induction was performed for 2.5 hours using 0.33 mM IPTG. After harvesting the bacterials, cells were resuspended in PBS-KMT (10 mM Na phosphate, pH 7.5, 130 mM NaCl, 3 mM KCl, 1 mM MgCl, 0.1% Tween-20), containing 1 mM 2-Mercaptoethanol, protease inhibitors and 1 mM Lysozyme, incubated for 30 min at RT and disrupted by sonification. The soluble supernatant after centrifugation was transferred to GSH sepharose column for purification. After washing the column with 10 column volumes of 20 mM Hepes, pH 7.5, 150 mM NaCl the GST fusion protein was eluted with 20 mM GSH and analyzed on a SDS gel to confirm expression.

4. Peptide Generation by Removal of GST Tag by Cleavage with PreScission® Protease

[0103] An example for PreScission® cleavage of one peptide from the GST-peptide fusion is shown below. The GST-tag was removed by incubation with PreScission® Protease (Amersham Pharmacia): 2.5 mg of fusion protein was incubated with 160 U PreScission® and digested for 16 hours at 5° C. on the sealed GSTrap FF column containing the bound GST fusion protein. After the overnight incubation a second GSTrap FF column was connected to remove the GST-tagged protease PreScission®. The sample was applied with a flow rate of 0.2 ml/min, the flow through was collected in small aliquot samples and analyzed by SDS gel electrophoresis and the amount of peptide was calculated by OD₂₈₀ measurement (ca. 700 μg).

Example 3

Affinity Purification of PT

A. Analysis of Fermentation Supernatant on Denaturing Gels

[0104] Two process fluids were considered as potential starting material for affinity chromatography process:

TABLE-US-00009 Sample A Concentrated culture filtrate containing 10-50 μg/ml (~0.09-0.45 μM) crude PT, fermentation supernatant Sample B Absorption chromatography supernatant containing 9-45 μg/ml (~0.08-0.4 μM) crude PT

To visualize the complexity of these process fluids, both samples were analyzed by denaturating polyacrylamid gelelectrophoresis. Mainly high molecular weight components of sample A are removed by the absorption chromatography (sample B).

B. Immobilization of Synthetic Biotinylated Core Peptides to Streptavidin Sepharose and Verification of Binding to Purified PT

1. Peptide Immobilization to Streptavidin Sepharose

[0105] For binding of biotinylated peptides to streptavidin sepharose (Amersham High Performance 71-5004-40), 200 μl of 50% slurry of pre-washed streptavidin sepharose were incubated with 1 nmol peptide (10 μl of 100 μM peptide solution) in 1 ml HEPES buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.025% TritonX-100) at 4° C. Under the applied conditions the high binding capacity of streptavidin sepharose should allow immobilization of 100% of the biotinylated peptide (10 pmol peptide per μl packed sepharose).

2. Binding of Purified PT to Immobilized Peptides

[0106] 200 μl of sepharose loaded with peptides (10% ige slurry, containing immobilized ˜200 pmol peptide) were transferred to a Mobicol column (MoBiTec, 10 μm filter) and the supernatant was removed by centrifugation for 1 min at 2000 rpm. After 4 washes with HEPES buffer, the sepharose was resuspended in 200 μl HEPES buffer containing 100 pmol purified Pertussis Toxin and incubated on a rotating wheel for 1 hour at room temperature. The unbound fraction was separated by centrifugation (supernatant after binding; applied to gel analysis). Subsequently the peptide-streptavidin sepharose was washed three-times with cold HEPES buffer (each 200 μl) and resuspended in 20 μl loading buffer (30 mM Tris, pH 6.8, 1% SDS, 1% β-Mercaptoethanol, 12.5% Glycerol, 0.005% Bromphenol Blue) to elute bound PT. After 5 min incubation at 95° C. the loading buffer was collected by centrifugation and subsequently used for gel analysis (FIG. 15). As a control streptavidin sepharose without peptide was contacted with PT under identical conditions. Under the applied conditions the Pertussis toxin peptide binder clones pp26 5n, 5c, 9n, 15n and the gurmarin clones 10n, 19n, 15n, 9n show a clear binding to purified PT. All positive binder candidates were able to bind the intact hexameric PT.

C. Immobilization of Synthetic Biotinylated Core Peptides to Streptavidin Sepharose and Verification of Binding to PT Out of the Fermentation Supernatant.

[0107] Peptide immobilization to streptavidin sepharose and binding analysis to PT out of fermentation supernatants was performed as described in chapter 0 with the exception that the peptide streptavidin sepharoses were incubated with 200 μl Sample A (fermentation supernatant) or with 200 μl Sample B (absorption chromatography supernatant column, see chapter 0) and were subsequently washed 4-times with HEPES buffer at RT. The results of the binding analysis is presented in FIG. 16. Under the applied conditions the Pertussis toxin pp26 binder clones 9n, 15n and the gurmarin clones 9n, 15n were able to bind very efficiently the intact PT hexamer out of the fermentation supernatants Sample A and Sample B. Note that under the applied conditions the pp26 binder clone 5 and the gurmarin binder clones 10 and 19 might bind PT with lower affinity. Although the PT binding to these peptides out of Sample A and Sample B were not detected under the conditions, these binders might be still qualified for application as ligand in an affinity chromatography column (a column allows retention of PT by rebinding effects and therefore would minimize the k_off problematic).

D. Thermodynamic Data on Immobilized Peptides

[0108] For estimation of peptides binding capacity 20 pmol of sepharose-immobilized peptides were incubated with an excess of 100 pmol PT in a volume of 200 μl HEPES buffer (corresponds to 500 nM PT). After washing, the fraction of peptide-streptavidin sepharose bound PT was quantified by gel analysis. This allows directly to calculate the fraction of binding active peptide under the applied conditions (assuming the PT/peptide binding ratio is 1:1). Under the assumption that a concentration of 500 nM PT is high enough to reach B_max for all peptides. The results of the analysis are shown in Table 7. The values presented therein are estimations for the expectable binding capacities of the peptides. An exact evaluation of binding capacity (B_max) and dissociation constant (K_D) of the most suitable binder may also be performed.

TABLE-US-00010 TABLE 7 Overview about fraction of binding active peptides under the applied experimental conditions Peptide name pp26 pp26 pp26 gurm gurm gurm gurm 5n 9n 15n 9n 10n 15n 19n Fraction of >5%¹ >50% >12.5% >12.5% |>5%¹ >50% >5%¹ binding active peptide ¹Calculation difficult because signals were near the detection limit

E. Analysis of the Stability of the Purified Pertussis Toxin Hexamer Under Defined Buffer Conditions (pH, Salt Concentration, Detergents), Using Acceptable Quality Grade Raw Materials Versus Health Authorities Requirements

[0109] The Pertussis toxin hexamer stability was tested under a broad range of pH and salt conditions on a BIAcore 2000 instrument. For this purpose 2000 RU of biotinylated PT were loaded on a streptavidin chip. Subsequently different buffers were applied to the chip immobilized PT for 2 min with a flow rate of 30 μl/min. After the end of each buffer injection the chip was equilibrated with HBS/EP running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl 3 mM EDTA 0.005% polysorbate 20 (v/v), at least 2 min with a flowrate of 30 μl/min). The difference of the measured RU signal before and after buffer injection correlates to the reduction of PT hexamer on the chip. This reduction was interpreted as loss of stability of PT hexamer under the applied buffer conditions.

[0110] The analyzed pH range was between pH 2 and 10.5 using the following buffers: 10 mM glycine buffer (BIAcore, pH 2; 2.5; 3), 10 mM acetate buffer (BIAcore, pH 4; 4.5; 5; 5.5), 50 mM Tris/HCl (pH 8.5) and 100 mM carbonate buffer (pH 9.6 and 10.5). BIAcore sensorgrams demonstrating the influence of the pH on the PT hexamer stability were generated, and the results of the BIAcore analysis are summarized in Table 8. Under the applied conditions, Pertussis toxin hexamer was shown to be stable over a broad pH range between pH 2.5-10.5.

TABLE-US-00011 TABLE 8 Pertussis toxin hexamer stability under different pH conditions. pH 2 2.5 3 4 4.5 5 5.5 8.5 9.6 10.5 PT 93 98 100 100 100 100 100 100 98 95 hexa- mer stability (%)

[0111] The influence of different salt conditions on the PT hexamer stability were investigated in comparable experiments on the BIAcore 2000 instrument for NaCl, KCl and MgCl₂ at pH 5.0 (10 mM acetate buffer) and pH 8.5 (50 mM Tris/HCl) respectively. An overview about PT hexamer stability under the applied salt conditions is shown in Table 9. The hexamer was stable in buffer (at pH 5 and 8.5) containing up to 2.5 M NaCl or up to 2 M KCl. In case of MgCl₂ the PT hexamer was stable in a buffer containing up to 2 M MgCl₂ at pH 8.5.

TABLE-US-00012 TABLE 9 Pertussis toxin hexamer stability under different salt conditions PT hexamer stability pH 5 pH 8.5 NaCl 0-2.5M 0-2.5M stable Stable KCl 0-2.0M 0-2.0M stable Stable MgCl₂ Nd 0-2.0M Stable

F. Establish Defined Wash and Elution Conditions Allowing a Specific Affinity Purification of PT Out of Fermentation Supernatant (pH, Salt Concentration, Detergents).

[0112] After the determination under which conditions the Pertussis toxin hexamer is stable, the next step was to investigate the wash and elution conditions for the bound Pertussis toxin to the immobilized peptides pp26 clone 9 and 15 and gurmarin clone 9 and 15.

1. Evaluation of PT/Peptide Stability Using the BIAcore 2000 Instrument

[0113] The stability of PT/peptide complexes were investigated using the BIAcore 2000 instrument under different pH and salt conditions that were shown before not to interfere with the PT hexamer stability. 500-1000 RU of the synthetic peptides were immobilized on BIAcore streptavidin chips. To allow binding of PT to the immobilized peptides, 20 nM purified PT in HEPES buffer was injected for 1 minute. After equilibration with HBS/EP running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl 3 mM EDTA 0.005% polysorbate 20 (v/v)) the PT/peptide complexes were washed by injection of

[0114] (a) 100 mM carbonate buffer at pH 10.5 and 9.5

[0115] (b) 10 mM acetate buffer at pH 5.5, 5.0, 4.5, and 4.0

[0116] (c) 10 mM glycine buffer at pH 3.0 and 2.5

[0117] (d) 0.5, 1.0, 1.5, 2.0 M NaCl in 10 mM acetate buffer, pH 6.0,

[0118] (e) 0.5, 1.0, 1.5, 2.0 M NaCl in 50 mM Tris/HCl buffer, pH 8.5,

[0119] (f) 0.5, 1.0, 1.5, 2.0 M KCl in 10 mM acetate buffer, pH 6.0,

[0120] (g) 0.5, 1.0, 1.5, 2.0 M KCl in 50 mM Tris/HCl buffer, pH 8.5,

[0121] (h) 0.5, 1.0, 1.5, 2.0 M NaCl in 10 mM acetate buffer, pH 6.0,

[0122] (i) 0.5, 1.0, 1.5, 2.0 M NaCl in 50 mM Tris/HCl buffer, pH 8.5.

[0123] After the end of each buffer injection, the chip was equilibrated with HBS/EP running buffer. The loss of PT hexamer on the chip under the applied buffer conditions (difference of measured RU signal before and after buffer injection) reflects the PT/peptide complex stability. An overview about the pH range stability and salt stability of all PT/peptide complexes is summarised in Table 10. All of the PT/peptide complexes were completely destabilized in the presence of 100 mM carbonate, pH 10.5 as well as 10 mM glycine, pH 2.5. For gurmarin peptide 9, buffers containing 2.5 M NaCl or at least 0.5 M MgCl₂ interfere with PT/peptide complex stability. PT complexes with gurmarin peptide 15 were additionally destabilized in the presence of at least 1.5 M MgCl₂ in 50 mM Tris/HCl, pH 8.5).

TABLE-US-00013 TABLE 10 Effect of different pH and salt conditions on the stability of the PT/peptide complexes pp26 peptide 9 pp26 peptide 15 gurmarin peptide 9 gurmarin peptide 15 pH range 3-9 3-9 3-9 3-9 stability of stable, stable, stable, stable, the complex instable at pH 2.5 instable at pH 2.5 or instable at pH 2.5 or instable at pH 2.5 or or 10.5 10.5 10.5 10.5 pH 6 pH 8.5 pH 6 pH 8.5 pH 8.5 pH 6 pH 8.5 NaCl 2M 2M 2M 2M strong sensitive to salt sensitive to salt stability of stable stable stable stable the complex KCl 2M 2M 2M 2M strong sensitive to salt sensitive to salt stability of stable stable stable stable the complex MgCl₂ stable Sensitive stable up Elution ≧1.5, Elution ≧0.5M Elution ≧1M, Elution ≧1M, stability of from 125 mM, to 2 M but complete complete the complex complete not elution ≧2M elution ≧2M elution ≧2M complete

2. Evaluation of Wash Conditions for Purification of PT on Peptide Streptavidin Sepharose

[0124] Wash conditions were tried to apply close to the established conditions for pertussis toxin purification process on asialofetuin (washing with 50 mM Tris/HCl, pH 7.5, with or without 1 M NaCl). The Pertussis toxin purification protocol was optimized for the peptides pp26 clone 9 and 15 and gurmarin clone 9 and 15. 200 pmol of each peptide immobilized on 20 μl sepharose were incubated with 100 μl 50 mM Tris/HCl, pH 7.5 and 100 μl sample A or sample B to allow binding of PT. Subsequently the peptide sepharose with bound PT fraction was washed under 3 different conditions, as shown below:

[0125] (a) 3 times with 200 μl 50 mM Tris/HCl, pH 7.5;

[0126] (b) 3 times with 200 μl 50 mM acetate pH 6.0; and,

[0127] (c) 6 times with 200 μl 50 mM acetate pH 6.0.

[0128] After washing remaining material was eluted from the sepharose with 20 μl loading buffer (30 mM Tris, pH 6.8, 1% SDS, 1% β-Mercaptoethanol, 12.5% Glycerol, 0.005% Bromphenol Blue). All elutions were subsequently analyzed by PAGE on 12% Bis-Tris-Gels (MES running buffer) and silver staining (FIG. 17).

[0129] Washing with 50 mM acetate, pH 6.0, is more stringent and reduces the back ground of high molecular weight impurities more efficient than washing with 50 mM Tris/HCl, pH 7.5. But under these washing conditions the PT/peptide complexes are less stable, especially in case of the gurmarin peptide 9 and repeated washes with 50 mM acetate, pH 6.0 (6 washes). In contrast to 50 mM Tris/HCl, pH 7.5, the loss of peptide immobilized PT was more dramatic when washing with 50 mM acetate, pH 6.0 was repeated 10 to 20 times (as an example shown for pp26 peptide 9 in FIG. 18).

3. Evaluation of Elution Conditions for Purification of PT on Peptide Streptavidin Sepharose

[0130] Elution of PT from peptide sepharose was tested under conditions that are compatible with hexamer stability.

a. Elution by MgCl₂

[0131] As shown above by BIAcore 2000 measurements all PT/peptide complexes were sensitive against 2 M MgCl₂, conditions that were shown not to be critical for PT hexamer stability. The elution efficiencies of defined MgCl₂ concentrations were evaluated for PT that was bound on streptavidin sepharose via one of the four immobilized synthetic peptides. 400 pmol of each peptide immobilized on 20 μl sepharose were incubated with 100 μl 50 mM Tris/HCl, pH 7.5 and 100 μl sample A to allow binding of PT. After 4 washes with 50 mM Tris/HCl, pH 7.5 (200 μl each), the bound fraction of PT was eluted using 3 consecutive 20 μl volumes of

[0132] (a) 0.2 M MgCl₂ in 50 mM Tris/HCl, pH 8.5, or

[0133] (b) 0.5 M MgCl₂ in 50 mM Tris/HCl, pH 8.5, or

[0134] (c) 1.0 M MgCl₂ in 50 mM Tris/HCl, pH 8.5, or

[0135] (d) 1.5 M MgCl₂ in 50 mM Tris/HCl, pH 8.5, or

[0136] (e) 2.0 M MgCl₂ in 50 mM Tris/HCl, pH 8.5.

[0137] Remaining material was afterwards eluted from the peptide streptavidin sepharose with 20 μl loading buffer (30 mM Tris, pH 6.8, 1% SDS, 1% β-Mercaptoethanol, 12.5% Glycerol, 0.005% Bromphenol Blue). All elutions were analyzed by PAGE on 12% Bis-Tris-Gels (MES running buffer) and silver staining (FIG. 19). As shown in the experiment elution with MgCl₂ was more efficient for the gurmarin peptides than for the pp26 peptides although a substantial amount of PT still remained on the peptide streptavidin sepharose.

b. Elution by pH-Shift

[0138] The BIAcore measurements revealed that PT was elutable from all peptides with acidic (pH of 2.5) or basic (pH of 10.5) buffer conditions that were not critical for PT hexamer stability (50 mM glycine, pH 2.5 more gentle for PT hexamer stability than 100 mM carbonate buffer, pH 10.5). 200 pmol of each peptide immobilized on 20 μl sepharose were incubated with 100 μl 50 mM Tris/HCl, pH 7.5 and 100 μl sample A to allow binding of PT. After 4 washes with 50 mM Tris/HCl, pH 7.5 (200 μl each), PT was eluted from the peptide streptavidin sepharose by 3 consecutive 40 μl elutions with 50 mM glycine, pH 2.5, or 100 mM carbonate buffer, pH 10.5. Remaining material was subsequently eluted from the peptide streptavidin sepharose with 20 μl loading buffer (30 mM Tris, pH 6.8, 1% SDS, 1% β-Mercaptoethanol, 12.5% Glycerol, 0.005% Bromphenol Blue). All elutions were analyzed by PAGE on 12% Bis-Tris-Gels (MES running buffer) and silver staining (FIG. 20). Nearly all of PT was elutable from the peptide streptavidin sepharose using 50 mM glycine, pH 2.5 as well as using 100 mM carbonate buffer, pH 10.5.

4. Apply Optimized Conditions for Small Scale Purification Scheme, Confirm Binding Capacity

[0139] a. Purification of PT from Sample B Under Optimized Wash and Elution Conditions (4 μl Column)

[0140] Optimized wash and elution conditions were combined to allow the purification of PT on peptide streptavidin sepharoses out of Sample B. To reduce unspecific binding of PT the optimal peptide/streptavidin sepharose ratio was titrated for each peptide before. Subsequently the Sample B/peptide streptavidin sepharose ratio was optimized in respect to high recovery of PT per expectable high (moderate) input of peptide. These conditions were applied to the following small scale column purifications.

[0141] For pp26 peptide 9 and gurmarin peptide 15, the immobilization to streptavidin sepharose was performed by incubation of 16 μl streptavidin sepharose with 1600 pmol peptide. In case of pp26 peptide15, 16 μl streptavidin sepharose was incubated with 6000 pmol peptide (pp26/15 binds with lower efficiency to the streptavidin sepharose, might be explainable by incomplete peptide biotinylation). For gurmarin peptide 9, 8000 pmol were immobilized on 80 μl streptavidin sepharose. Subsequently the washed peptide streptavidin sepharoses were equally subdivided and transferred to 4 Mobilcom columns (with 10 μM filters).

[0142] Each column (containing 4 μl sepharose with 400 pmol peptide for pp26/9 and gur/15; 4 μl with undefined amount bound peptide pp26/15; 20 μl with 2000 pmol peptide for gur/9) was incubated with 400 μl Sample B (adjusted to pH 7.0-7.5 by addition of HCl) to allow binding of PT. After 5 washes with 50 mM Tris/HCl, pH 7.5 (each 100 μl), PT was eluted from the peptide streptavidin sepharose by consecutive elutions (3 elutions for pp26/9 and gur/15; 4 elutions for pp26/15 and gur/9), as follows:

[0143] (a) with 50 mM glycine, pH 2.5 (each 20 μl) in case of column 1, or

[0144] (b) with 100 mM carbonate buffer, pH 10.5 (each 20 μl) in case of column 2, or

[0145] (c) with 2 M MgCl₂ in 50 mM Tris, pH 8.5 (each 20 μl) in case of column 3. Remaining material on column 1-3 as well on column 4 was subsequently eluted from the peptide streptavidin sepharoses by elution with 20 μl loading buffer (30 mM Tris, pH 6.8, 1% SDS, 1% β-Mercaptoethanol, 12.5% Glycerol, 0.005% Bromphenol Blue). All elutions were analyzed by PAGE on 12% Bis-Tris-Gels (MES running buffer) and silver staining (FIGS. 21, 22). To calculate the yield of PT after purification on the peptide streptavidin sepharoses the pooled elutions 1-3 were analyzed by PAGE and silver staining and compared to defined amounts of purified PT separated on the same gel allowing an estimation (FIGS. 21B and 22B). Based on the gel estimation, the yield of purified PT was calculated as shown in Table 11.

TABLE-US-00014

[0145] TABLE 11 Calculation of the Pertussis Toxin yield after small scale column purification from sample B with pp26 peptide 9 or gurmarin peptide 15 as affinity ligands Total Yield relative to Yield relative to the Estimation from yield of input PT in amount of sepharose Peptide Elution with FIG. 13B and 14B PT sample B* bound peptide pp26 peptide 9 Glycine pH 2.5 2 pmol PT in 3/120 80 pmol >48% 20% of pooled elutions Carbonate pH 10.5 elution 2 pmol PT in 3/120 80 pmol >48% 20% of pooled elutions MgCl₂ 1 pmol PT in 6/120 20 pmol >12% 5% of pooled elutions gurmarin peptide 15 Glycine pH 2.5 2 pmol PT in 3/120 80 pmol >48% 20% of pooled elutions Carbonate pH 10.5 elution 2 pmol PT in 3/120 80 pmol >48% 20% of pooled elutions MgCl₂ 1 pmol PT in 3/120 40 pmol >24% 10% of pooled elutions *according to the documentation related to PT, the expected PT concentration of sample B is 9-45 μg/ml, corresponding to 0.8-0.41 pmol/μl. Calculation was performed as following: 400 μl of sample B × 0.41 pmol/μl = 164 pmol input PT

b. Determination of PT Yield During Affinity Purification Using Varying Peptide Densities on Streptavidin Sepharose

[0146] The PT binding to peptide streptavidin sepharose was investigated in dependence of varying concentration of peptide immobilized on the streptavidin sepharose as affinity ligand. For immobilization 1 μl volume of streptavidin sepharose was incubated with increasing amounts of peptide pp26/9 or gurmarin/15 (100, 200, 300, 400, 500, 1000 pmol peptide). Unbound fractions of peptides were removed from the sepharose by 3 washes with 50 mM Tris/HCl, pH 7.5 (on column). Subsequently each peptide straptavidin matrix was incubated with 600 μl Sample A to allow binding of PT. After 80 min each matrix was washed four times with 50 mM Tris/HCl, pH 7.5 (200 μl each) and subsequently eluted with 20 μl gel loading buffer (30 mM Tris, pH 6.8, 1% SDS, 1% β-Mercaptoethanol, 12.5% glycerol, 0.005% Bromphenol blue; incubation for 10 min at 95° C.). Elutions were analyzed by PAGE on 12% Bis-Tris-Gels (MES running buffer) and silver staining (FIG. 23). Amount of PT that was bound to peptide streptavidin sepharose was calculated by densitometric evaluation and plotted as a function of the amount of peptide initially used for immobilization to streptavidin sepharose (shown for pp26/9 in FIG. 23). A maximum of PT binding was reached when 300-400 pmol peptide were used for immobilization to 1 μl streptavidin sepharose. Higher amounts of peptide did not result in higher PT binding probably reflecting effects of steric hindrance of PT.

[0147] The effectively bound fraction of peptide (pp26/9 or gurmarin/15) when an input of 400 pmol peptide was used for immobilization to 1 μl streptavidin sepharose, was evaluated by PAGE on a 12% Bis-Tris-Gel (MES running buffer) and silver staining after elution with gel loading buffer (heating at 95° C. for 10 min). Amount of elutable peptide was estimated by direct comparison to defined amounts of purified PT on the same gel (data not shown): for pp26/9: 100-150 pmol; for gurmarin/15: 50 pmol.

c. Determination of PT Yield Using Varying Amounts of Sample B at Constant Concentration of Peptide Sepharose During Affinity Purification.

[0148] For peptide immobilization 400 pmol pp26/9 or gurmarin/15 were incubated with 1 μl streptavidin sepharose for 1 h at RT. The peptide sepharose was washed 3 times with 200 μl 50 mM Tris pH 7.5 buffer and subsequently incubated with varying amounts of Sample B (50, 66, 100, 200, 400, 600 μl, adjusted before to pH 7.0-7.5 by addition of HCl) for 1 hour at RT. The affinity matrices were washed 4 times with 100 μl 50 mM Tris/HCl, pH 7.5, and eluted by 4 consecutive elutions with 100 mM Carbonate buffer at pH 10.5 (each 20 μl). 5 μl of the pooled elutions (total 80 μl) were analyzed by PAGE on 12% Bis-Tris-Gels (MES running buffer) and silver staining. The amount of eluted PT was calculated on the basis of direct comparison to defined amounts of purified PT on the same gel as mass standard (FIG. 24, Table 12).

TABLE-US-00015 TABLE 12 Yield of PT Input PT Ratio Amount of PT relative to input (pmol) peptide:PT bound (pmol) amount of PT Input peptide 16K9 (pmol) 100 300 1:3 ~100 33% 100 200 1:2 ~88 44% 100 100 1:1 ~40 40% 100 50 2:1 ~24 48% 100 33.3 3:1 ~16 48% 100 25 4:1 ~24 96% Input peptide 17K15 (pmol) 100 300 1:3 ~80 27% 100 200 1:2 ~64 32% 100 100 1:1 ~56 56% 100 50 2:1 ~16 32% 100 33.3 3:1 ~16 32% 100 25 4:1 ~8 32% Input asiaolfetuin (pmol) 100 200 1:2 ~8 4% 100 100 1:1 ~16 16% 100 85.6 20:17 ~8 9% 100 50 2:1 ~8 16% 100 33.3 3:1 ~8 24% 100 25 4:1 ~8 35%

[0149] To compare the purification efficiencies of the peptide streptavidin sepharoses with asialofetuin sepharose a titration experiment with asialofetuin sepharose was performed in parallel under comparable conditions (same amount of affinity ligand per reaction immobilized on sepharose, corresponding to ˜100 pmol affinity ligand effectively bound to the sepharose). This was accomplished by incubation of 6.85 μl of asialofetuin sepharose (batch number FA 053198: density 1.1 mg/ml, 14.6 pmol/μl) with varying amounts of Sample B (50, 66, 100, 171.3, 200, 400 μl, adjusted before to pH 7.0-7.5 by addition of HCl) for 1 hour at RT. Subsequently the asialofetuin sepharose was washed and bound PT was eluted and analyzed as described above. The binding efficiency of peptide streptavidin sepharose under the applied purification conditions was significantly higher than the binding efficiency of asialofetuin sepharose.

d. Reutilization of Peptide Sepharose for Repeated PT Binding and Elution

[0150] To investigate the reusability of peptide loaded sepharose (pp26/9 and gurmarin/15) for repeated binding and elution of PT the sepharoses were applied for repeated cycles of PT binding, elution and regeneration (in total 4 times). For peptide immobilization 600 pmol pp26/9 or gurmarin/15 were incubated with 2 μl streptavidin sepharose over night at RT and subsequently washed 3 times with HEPES buffer. For binding of PT each peptide streptavidin sepharose was incubated with 400 μl sample B (adjusted to pH 7.0-7.5 by addition of HCl) for 1 hour at RT and washed 4 times with 50 mM Tris/HCl, pH 7.5 (each 200 μl). PT was eluted by 4 consecutive elutions with 100 mM Carbonate buffer at pH 10.5 (each 20 μl). Subsequently the column matrices were regenerated by three washes with 10 mM HCl (1×20 μl, 2×100 μl) and afterwards neutralized by two washes with 200 μl 50 mM Tris/HCl, pH 7.5. This binding, elution and regeneration procedure was applied to the peptide sepharose for three additional times. 4 μl of the pooled elutions (in total 80 μl) and 7 μl of the first regeneration buffer from each binding/elution/regeneration cycle were analyzed by PAGE on 12% Bis-Tris-Gels (MES running buffer) and silver stained, indicating that the peptide sepharose may be re-utilized. (FIG. 25).

5. Large-Scale FPLC-Purification of PT

[0151] Optimized conditions for PT binding and elution were applied for large scale FPLC purification (0.5 ml column), as shown below:

[0152] A) Immobilization of Biotinylated Peptide to Streptavidin-Sepharose:

[0153] 200 nmol peptide pp26/9 were incubated for 1 h 30 min at room temperature on a rotating wheel with 1 ml 50% Streptavidin-sepharose in volume of 10 ml (HEPES-buffer). After incubation the sepharose was washed 3× with 50 mM Tris pH 7.5.

[0154] B) Binding of PT (Out of Sample B):

[0155] The estimated amount of peptide effectively immobilized on 500 μl sepharose was 50 nmol. The peptide-sepharose was incubated with 25 ml sample B for 1 h 30 min at room temperature in a head over tail rotator (assumed concentration of PT ˜0.5 pmol/μ1, corresponding to 12.5 nmol in 25 ml, corresponding to a ratio of immobilized peptide to amount of PT of 4:1).

[0156] C) FPLC-Column:

[0157] After incubation the sepharose was transferred to a column (Pharmacia HR 5/5) During packing of the column the sepharose was washed with 50 mM Tris pH 7.5 (2-3 ml). Subsequently the column was taken in the flow path and washed with 20 column volumes (10 ml) 50 mM Tris ph 7.5. Immobilized PT was eluted with 11 ml 100 mM carbonate buffer pH 10.5. The elution fractions were collected in 500 μl fractions (Pharmacia Fraction Collector FRAC-100) and the elution profile was evaluated by measurement of the UV absorbance at 280 nm. After elution the column was washed with 1.5 ml 50 mM Tris pH 7.5 and subsequently regenerated with 2.5 ml 10 mM HCl followed by neutralization with 10 ml 50 mM Tris pH 7.5.

[0158] D) Analysis of Elution Fractions and Calculation of Yield:

[0159] The elution fractions were analyzed by PAGE (12% Bis-Tris-Gel, MES running buffer) and silver staining (FIG. 26). Concentration of PT was determined by measuring the absorbance of the elution fractions at 280 nm (A₂₈₀) and comparing these results with a calibration curve prepared with purified PT (see table in FIG. 26).

[0160] The amount of PT was additionally calculated on the basis of direct comparison to defined amounts of purified PT on the same gel as mass standard. Gel estimation leads to a yield of 8100 pmol PT. This correlates very well with the concentration determination using A₂₈₀. If it is assumed that 25 ml sample B contains 1125 μg of PT, more than 69%-72% is eluted of PT under these conditions. This result was verified by repetition of the FPLC run using the same peptide-sepharose after regeneration to bind PT out of 25 ml sample B. In this experiment, 803 μg PT was purified (A₂₈₀) (Table 13).

TABLE-US-00016 TABLE 13 Determination of concentration of PT in elution fraction (FPLC run #2) using A₂₈₀ A₂₈₀ μg/ml Elu1 0 0 Elu2 0 0 Elu3 0.091 85 Elu4 0.4185 391 Elu5 0.354 331 Elu6 0.2835 265 Elu7 0.212 198 Elu8 0.148 138 Elu9 0.0975 91 Elu10 0.0585 55 Elu11 0.0315 29 Elu12 0.025 23 Total 3-12 = 803 μg

TABLE-US-00017 TABLE 14 Summary of PT Purification Results Yield PT in Relative Yield versus 12x 0.5 ml input amount of PT fractions (1125 μg in 25 ml) (6 ml) (pmol/pmol or μg/μg) Purity 1. purification 772-813 μg 69%-72% Comparable to PT run purified on asialofetuin sepharose, 100% 2. purification 803 μg 71% Comparable to PT run purified on asialofetuin sepharose, 100%

6. Evaluation of Equilibrium and Rate Constants of the pp26 Peptide 9/Pertussis Toxin Complex Formation

[0161] Equilibrium constants and rate constants for the pp26 K9/PT complex formation were evaluated using the BIAcore 2000 instrument in HBS/EP running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% (v/v) polysorbate 20) at room temperature. Binding of varying concentrations of pp26-K9 (concentrations between 2.5 nM and 100 nM) to PT immobilzed on a CM5 chip (immobilization of 6000 RU via amine coupling method) were analyzed at a flow rate of 30 μl/min. Quantitative elution of PT bound peptides were obtained by using 3 mM HCl, pH 2.5. Deducible equilibrium and rate constants were analyzed using the BIAevaluation software, the results of which are shown below:

[0162] Dissociation equilibrium constant K_D→7.5×10^-9 M

[0163] Association equilibrium constant K_A→1.3×10^-8 M^-1

[0164] Association rate constant k_on→1.3×10⁵ M^-1×s^-1

[0165] Dissociation rate constant k_off→10^-3 s^-1

[0166] While the present invention has been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that come within the scope of the invention as claimed.

Sequence CWU 1

1

41517PRTArtificial SequenceSynthetic 1Asn Gly Ser Phe Ser Gly Phe 1 5 27PRTArtificial SequenceSynthetic 2Asn Gly Ser Phe Ser Gly Cys 1 5 37PRTArtificial SequenceSynthetic 3Asp Gly Ser Phe Ser Gly Phe 1 5 47PRTArtificial SequenceSynthetic 4Xaa Gly Ser Phe Ser Gly Xaa 1 5 530PRTArtificial SequenceSynthetic 5Arg Ser Ser His Cys Arg His Arg Asn Cys His Thr Ile Thr Arg Gly 1 5 10 15 Asn Met Arg Ile Glu Thr Pro Asn Asn Ile Arg Lys Asp Ala 20 25 30 629PRTArtificial SequenceSynthetic 6Ser Thr Met Asn Thr Asn Arg Met Asp Ile Gln Arg Leu Met Thr Asn 1 5 10 15 His Val Lys Arg Asp Ser Ser Pro Gly Ser Ile Asp Ala 20 25 730PRTArtificial SequenceSynthetic 7Arg Ser Asn Val Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp 1 5 10 15 Asp Arg Pro His Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala 20 25 30 830PRTArtificial SequenceSynthetic 8Arg Ser Trp Arg Asp Thr Arg Lys Leu His Met Arg His Tyr Phe Pro 1 5 10 15 Leu Ala Ile Asp Ser Tyr Trp Asp His Thr Leu Arg Asp Ala 20 25 30 934PRTArtificial SequenceSynthetic 9Ser Gly Cys Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val 1 5 10 15 Cys Cys Glu Pro Leu Glu Cys Ile Tyr Thr Ser Glu Leu Tyr Ala Thr 20 25 30 Cys Gly 1034PRTArtificial SequenceSynthetic 10Ser Gly Cys Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Val Asp Glu 1 5 10 15 Cys Cys Glu Pro Leu Glu Cys Phe Gln Met Gly His Gly Phe Lys Arg 20 25 30 Cys Gly 1135PRTArtificial SequenceSynthetic 11Ser Gly Cys Val Lys Lys Asp Glu Leu Cys Ser Gln Ser Val Pro Met 1 5 10 15 Cys Cys Glu Pro Leu Glu Cys Lys Trp Phe Asn Glu Asn Tyr Gly Ile 20 25 30 Cys Gly Ser 35 1234PRTArtificial SequenceSynthetic 12Ser Gly Cys Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Glu 1 5 10 15 Cys Cys Glu Pro Leu Glu Cys Thr Lys Gly Asp Leu Gly Phe Arg Lys 20 25 30 Cys Gly 1335PRTArtificial SequenceSynthetic 13Gln Gln Cys Val Lys Lys Asp Glu Leu Cys Ile Pro Tyr Tyr Leu Asp 1 5 10 15 Cys Cys Glu Pro Leu Glu Cys Lys Lys Val Asn Trp Trp Asp His Lys 20 25 30 Cys Ile Gly 35 1431PRTArtificial SequenceSynthetic 14Cys Val Lys Lys Asp Glu Leu Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys 1 5 10 15 Glu Pro Leu Glu Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 20 25 30 15141DNAArtificial SequenceSynthetic 15agtggctcaa gctcaggatc aggctgcgtc aagaaagacg agctctgcnn snnsnnsnns 60nnsnnstgct gtgagcccct cgagtgcnns nnsnnsnnsn nsnnsnnsnn snnstgcggc 120agcggcagtt ctgggtctag c 1411684DNAArtificial SequenceSynthetic 16taatacgact cactataggg acaattacta tttacaatta caatgcacca tcaccatcac 60catagtggct caagctcagg atca 841744DNAArtificial SequenceSynthetic 17ttttaaatag cggatgctac taggctagac ccagaactgc cgct 441810RNAArtificial SequenceSynthetic 18uagcggaugc 101953PRTArtificial sequenceSynthetic 19Thr Met Val Met Gly Arg Gly Ser His His His His His His Ala Arg 1 5 10 15 Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Ala Asn Ala Pro 35 40 45 Lys Ala Ser Ala Ile 50 206PRTArtificial SequenceSynthetic 20His His His His His His 1 5 216PRTArtificial SequenceSynthetic 21Asp Ala Asn Ala Pro Lys 1 5 22127DNAArtificial sequenceSynthetic 22agcggatgcc ttcggagcgt tagcgtcsnn snnsnnsnns nnsnnsnnsn nsnnsnnsnn 60snnsnnsnns nnsnnsnnsn nsnnsnnsnn snnsnnsnns nnsnnagatc tagcatgatg 120atgatga 1272381DNAArtificial SequenceSynthetic 23taatacgact catagggaca attactattt acaattacaa tgggacgtgg ctcacatcat 60catcatcatc atgctagatc t 812432DNAArtificial SequenceSynthetic 24aattaaatag cggatgcctt cggagcgtta gc 322518DNAArtificial SequenceSynthetic 25tgtaaaacga cggccagt 182654PRTArtificial SequenceSynthetic 26Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Gly Ser Val Gly His Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg Arg Phe Leu Asn Leu Arg Trp Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 2754PRTArtificial SequenceSynthetic 27Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ile Val Met Arg Ala Pro Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg Arg Tyr Met Leu Lys His Met Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 2854PRTArtificial SequenceSynthetic 28Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Lys Ala Phe Arg Tyr Ser Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg Lys Trp Leu Lys Ala Arg Phe Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 2954PRTArtificial SequenceSynthetic 29Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Leu Arg Ser Ser Ile Asp Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Tyr Lys Trp Met Gln Arg Arg Leu Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 3054PRTArtificial SequenceSynthetic 30Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Trp Pro Arg Arg His Lys Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Leu Glu Met Leu Glu Arg Lys Arg Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 3153PRTArtificial SequenceSynthetic 31Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Met Ser Met Ala Cys Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Lys Tyr His Gly Tyr Phe Trp Leu Cys Gly Ser Gly 35 40 45 Ser Ser Gly Ser Ser 50 3254PRTArtificial SequenceSynthetic 32Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Val Trp Phe Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Thr Tyr Gln Ser Gly Tyr Tyr Trp Leu Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 3354PRTArtificial SequenceSynthetic 33Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Pro Trp Tyr Trp Arg Cys Cys Glu 20 25 30 Pro Leu Glu Cys Val Tyr Thr Ser Gly Tyr Tyr Tyr Ser Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 3454PRTArtificial SequenceSynthetic 34Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Ile Tyr Thr Ser Glu Leu Tyr Ala Thr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 3554PRTArtificial SequenceSynthetic 35Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Val Phe Tyr Phe Pro Asn Cys Cys Glu 20 25 30 Pro Leu Glu Cys Arg Trp Val Asn Asp Asn Tyr Gly Trp Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 3653PRTArtificial SequenceSynthetic 36Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Met Ser Met Ala Cys Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Lys Tyr His Gly Tyr Phe Trp Leu Cys Gly Ser Gly 35 40 45 Ser Ser Gly Ser Ser 50 3754PRTArtificial SequenceSynthetic 37Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Thr Thr Ala Ser Lys Ser Cys Cys Glu 20 25 30 Pro Leu Glu Cys Lys Trp Thr Asn Glu His Phe Gly Thr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 3854PRTArtificial SequenceSynthetic 38Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ser Gln Ser Val Pro Met Cys Cys Glu 20 25 30 Pro Leu Glu Cys Lys Trp Phe Asn Glu Asn Tyr Gly Ile Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 3954PRTArtificial SequenceSynthetic 39Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Ile Tyr Thr Ser Glu Leu Tyr Ala Thr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 4054PRTArtificial SequenceSynthetic 40Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 4153PRTArtificial SequenceSynthetic 41Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Met Trp Ser Arg Glu Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Tyr Tyr Thr Gly Trp Tyr Trp Ala Cys Gly Ser Gly 35 40 45 Ser Ser Gly Ser Ser 50 4254PRTArtificial SequenceSynthetic 42Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Val Asp Glu Cys Cys Glu 20 25 30 Pro Leu Glu Cys Phe Gln Met Gly His Gly Phe Lys Arg Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 4354PRTArtificial SequenceSynthetic 43Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Val Asp Glu Cys Cys Glu 20 25 30 Pro Leu Glu Cys Thr Lys Gly Asp Leu Gly Phe Arg Lys Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 4454PRTArtificial SequenceSynthetic 44Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 4554PRTArtificial SequenceSynthetic 45Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 4654PRTArtificial SequenceSynthetic 46Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Asn Trp Val Thr Pro Met Arg Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 4750PRTArtificial SequenceSynthetic 47Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asp Trp 1 5 10 15 Glu Leu Ser Pro Pro His Val Ala Ile Thr Thr Arg His Leu Ile Asn 20 25 30 Cys Thr Asp Gly Pro Leu Leu Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 4850PRTArtificial SequenceSynthetic 48Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Asn 1 5 10 15 Gly Glu Ser Thr Ser Asn Ile Leu Thr Thr Ser Arg Lys Val Thr Glu 20 25 30 Trp Thr Gly Tyr Thr Ala Ser Val Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 4950PRTArtificial SequenceSynthetic 49Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Gln Val 1 5 10 15 Thr Trp His His Leu Ala Asp Thr Val Thr Thr Lys Asn Arg Lys Cys 20 25 30 Thr Asp Ser Tyr Ile Gly Trp Asn Xaa Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 5050PRTArtificial SequenceSynthetic 50Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ile Ile 1 5 10 15 Val Ile His Asn Ala Ile Gln Thr His Thr Pro His Gln Val Ser Ile 20 25 30 Trp Cys Pro Pro Lys His Asn Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 5150PRTArtificial SequenceSynthetic 51Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ser His 1 5 10 15 Cys Arg His Arg Asn Cys His Thr Ile Thr Arg Gly Asn Met Arg Ile 20 25 30 Glu Thr Pro Asn Asn Ile Arg Lys Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 5250PRTArtificial SequenceSynthetic 52Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Thr Met 1 5 10 15 Asn Thr Asn Arg Met Asp Ile Gln Arg Leu Met Thr Asn His Val Lys 20 25 30 Arg Asp Ser Ser Pro Gly Ser Ile Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 5350PRTArtificial SequenceSynthetic 53Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Ser 1 5 10 15 Ala Leu Arg Arg Thr Glu Arg Thr Trp Asn Thr Ile His Gln Gly His 20 25 30 His Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 5450PRTArtificial SequenceSynthetic 54Met Gly Arg Gly Ser His

His His His His His Ala Arg Ser Trp Thr 1 5 10 15 Ser Met Gln Gly Glu Thr Leu Trp Arg Thr Asp Arg Leu Ala Thr Thr 20 25 30 Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 5550PRTArtificial SequenceSynthetic 55Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Val 1 5 10 15 Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 5649PRTArtificial SequenceSynthetic 56Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Cys Leu 1 5 10 15 Ala Thr Arg Asn Gly Phe Val Met Asn Thr Asp Arg Gly Thr Tyr Val 20 25 30 Lys Arg Pro Thr Val Leu Gln Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 5750PRTArtificial SequenceSynthetic 57Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Gly 1 5 10 15 Leu Ser Gly Thr Gln Thr Trp Lys Ile Thr Lys Leu Ala Thr Arg Leu 20 25 30 His His Pro Glu Phe Glu Thr Asn Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 5850PRTArtificial SequenceSynthetic 58Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Arg 1 5 10 15 Trp His Asn Trp Gly Leu Ser Asp Thr Val Ala Ser His Pro Asp Ala 20 25 30 Ser Asn Ser Leu Asn Met Met Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Asn 50 5949PRTArtificial SequenceSynthetic 59Met Gly Arg Gly Ser His His His His His His Leu Asp Leu Trp Gly 1 5 10 15 Pro Pro Ser Gly Ser Pro Arg Thr Arg Ser Thr Thr Gly Thr Ser Thr 20 25 30 Thr Ser Ser Pro Ser Thr Pro Gly Thr Leu Thr Leu Arg Arg His Pro 35 40 45 His 6049PRTArtificial SequenceSynthetic 60Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Gln 1 5 10 15 Pro Glu Val Lys Met Ser Ser Leu Val Asp Thr Ser Gln Thr Val Gly 20 25 30 Ala Ala Val Glu Thr Arg Thr Thr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 6150PRTArtificial SequenceSynthetic 61Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Arg 1 5 10 15 Asp Thr Arg Lys Leu His Met Arg His Tyr Phe Pro Leu Ala Ile Asp 20 25 30 Ser Tyr Trp Asp His Thr Leu Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 6250PRTArtificial SequenceSynthetic 62Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Thr 1 5 10 15 Ser Met Gln Gly Glu Thr Leu Trp Arg Thr Asp Arg Leu Ala Thr Thr 20 25 30 Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 6350PRTArtificial SequenceSynthetic 63Met Gly Arg Gly Ser His His His His His His His Ala Arg Ser Pro 1 5 10 15 Leu Trp Tyr His Tyr Asn Cys Trp Asp Thr Ile Cys Leu Ala Asp Trp 20 25 30 Leu Lys Asp Arg Pro His Gly Val Tyr Asp Ala Asn Ala Pro Lys Ala 35 40 45 Ser Ala 50 6450PRTArtificial SequenceSynthetic 64Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 6550PRTArtificial SequenceSynthetic 65Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 6650PRTArtificial SequenceSynthetic 66Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Val 1 5 10 15 Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 6731PRTArtificial SequenceSynthetic 67Arg Ser Ser His Cys Arg His Arg Asn Cys His Thr Ile Thr Arg Gly 1 5 10 15 Asn Met Arg Ile Glu Thr Pro Asn Asn Ile Arg Lys Asp Ala Lys 20 25 30 6830PRTArtificial SequenceSynthetic 68Arg Ser Ser His Cys Arg His Arg Asn Cys His Thr Ile Thr Arg Gly 1 5 10 15 Asn Met Arg Ile Glu Thr Pro Asn Asn Ile Arg Lys Asp Ala 20 25 30 6930PRTArtificial SequenceSynthetic 69Arg Ser Thr Met Asn Thr Asn Arg Met Asp Ile Gln Arg Leu Met Thr 1 5 10 15 Asn His Val Lys Arg Asp Ser Ser Pro Gly Ser Ile Asp Ala 20 25 30 7030PRTArtificial SequenceSynthetic 70Arg Ser Asn Val Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp 1 5 10 15 Asp Arg Pro His Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala 20 25 30 7130PRTArtificial SequenceSynthetic 71Arg Ser Trp Arg Asp Thr Arg Lys Leu His Met Arg His Tyr Phe Pro 1 5 10 15 Leu Ala Ile Asp Ser Tyr Trp Asp His Thr Leu Arg Asp Ala 20 25 30 7235PRTArtificial SequenceSynthetic 72Ser Gly Cys Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val 1 5 10 15 Cys Cys Glu Pro Leu Glu Cys Ile Tyr Thr Ser Glu Leu Tyr Ala Thr 20 25 30 Cys Gly Lys 35 7334PRTArtificial SequenceSynthetic 73Ser Gly Cys Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val 1 5 10 15 Cys Cys Glu Pro Leu Glu Cys Ile Tyr Thr Ser Glu Leu Tyr Ala Thr 20 25 30 Cys Gly 7434PRTArtificial SequenceSynthetic 74Ser Gly Cys Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Val Asp Glu 1 5 10 15 Cys Cys Glu Pro Leu Glu Cys Phe Gln Met Gly His Gly Phe Lys Arg 20 25 30 Cys Gly 7535PRTArtificial SequenceSynthetic 75Ser Gly Cys Val Lys Lys Asp Glu Leu Cys Ser Gln Ser Val Pro Met 1 5 10 15 Cys Cys Glu Pro Leu Glu Cys Lys Trp Phe Asn Glu Asn Tyr Gly Ile 20 25 30 Cys Gly Ser 35 7634PRTArtificial SequenceSynthetic 76Ser Gly Cys Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Glu 1 5 10 15 Cys Cys Glu Pro Leu Glu Cys Thr Lys Gly Asp Leu Gly Phe Arg Lys 20 25 30 Cys Gly 7728DNAArtificial SequenceSynthetic 77catgccatgg gacgtggctc acatcatc 287836DNAArtificial SequenceSynthetic 78gggttaaata gcggatgcct tcggagcgtt agcgtc 367938DNAArtificial SequenceSynthetic 79ggagatctca tatgcaccat caccatcacc atagtggc 388026DNAArtificial SequenceSynthetic 80gggttaaata gcggatgcta ctaggc 268160PRTArtificial SequenceSynthetic 81Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Gly Ser Val Gly His Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg Arg Phe Leu Asn Leu Arg Trp Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 8260PRTArtificial SequenceSynthetic 82Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ile Val Met Arg Ala Pro Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg Arg Tyr Met Leu Lys His Met Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 8360PRTArtificial SequenceSynthetic 83Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Lys Ala Phe Arg Tyr Ser Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg Lys Trp Leu Lys Ala Arg Phe Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 8458PRTArtificial SequenceSynthetic 84Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Leu Arg Ser Ser Ile Asp Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Tyr Lys Trp Met Gln Arg Arg Leu Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser 50 55 8560PRTArtificial SequenceSynthetic 85Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Trp Pro Arg Arg His Lys Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Leu Glu Met Leu Glu Arg Lys Arg Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 8658PRTArtificial SequenceSynthetic 86Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Met Ser Met Ala Cys Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Lys Tyr His Gly Tyr Phe Trp Leu Cys Gly Ser Gly 35 40 45 Ser Ser Gly Pro Ser Ser Ile Arg Tyr Leu 50 55 8760PRTArtificial SequenceSynthetic 87Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Val Trp Phe Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Thr Tyr Gln Ser Gly Tyr Tyr Trp Leu Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 8860PRTArtificial SequenceSynthetic 88Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Pro Trp Tyr Trp Arg Cys Cys Glu 20 25 30 Pro Leu Glu Cys Val Tyr Thr Ser Gly Tyr Tyr Tyr Ser Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 8955PRTArtificial SequenceSynthetic 89Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Ile Tyr Thr Ser Glu Leu Tyr Ala Thr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Gly 50 55 9060PRTArtificial SequenceSynthetic 90Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Val Phe Tyr Phe Pro Asn Cys Cys Glu 20 25 30 Pro Leu Glu Cys Arg Trp Val Asn Asp Asn Tyr Gly Trp Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 9159PRTArtificial SequenceSynthetic 91Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Met Ser Met Ala Cys Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Lys Tyr His Gly Tyr Phe Trp Leu Cys Gly Ser Gly 35 40 45 Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 9260PRTArtificial SequenceSynthetic 92Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Thr Thr Ala Ser Lys Ser Cys Cys Glu 20 25 30 Pro Leu Glu Cys Lys Trp Thr Asn Glu His Phe Gly Thr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 9360PRTArtificial SequenceSynthetic 93Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ser Gln Ser Val Pro Met Cys Cys Glu 20 25 30 Pro Leu Glu Cys Lys Trp Phe Asn Glu Asn Tyr Gly Ile Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 9460PRTArtificial SequenceSynthetic 94Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Ile Tyr Thr Ser Glu Leu Tyr Ala Thr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 9560PRTArtificial SequenceSynthetic 95Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 9659PRTArtificial SequenceSynthetic 96Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Met Trp Ser Arg Glu Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Tyr Tyr Thr Gly Trp Tyr Trp Ala Cys Gly Ser Gly 35 40 45 Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 9757PRTArtificial SequenceSynthetic 97Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Val Asp Glu Cys Cys Glu 20 25 30 Pro Leu Glu Cys Phe Gln Met Gly His Gly Phe Lys Arg Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Gly Ser Asn 50 55 9860PRTArtificial SequenceSynthetic 98Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Gly Leu Cys Trp Pro Arg Arg His Lys Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Leu Glu Met Leu Glu Arg Lys Arg Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 9960PRTArtificial SequenceSynthetic 99Met His His His His His His

Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Val Asp Glu Cys Cys Glu 20 25 30 Pro Leu Glu Cys Thr Lys Gly Asp Leu Gly Phe Arg Lys Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 10060PRTArtificial SequenceSynthetic 100Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 10160PRTArtificial SequenceSynthetic 101Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Asn Trp Val Thr Pro Met Arg Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 10262PRTArtificial SequenceSynthetic 102Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 10362PRTArtificial SequenceSynthetic 103Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Tyr Ala Ser Ala Ile 50 55 60 10450PRTArtificial SequenceSynthetic 104Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ile 50 10562PRTArtificial SequenceSynthetic 105Met His His His His His His Ser Asp Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 10662PRTArtificial SequenceSynthetic 106Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Arg Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 10752PRTArtificial SequenceSynthetic 107Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly 50 10849PRTArtificial SequenceSynthetic 108Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly 10958PRTArtificial SequenceSynthetic 109Leu His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Asp Pro 50 55 11050PRTArtificial SequenceSynthetic 110Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Asn 50 11146PRTArtificial SequenceSynthetic 111Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys 35 40 45 11254PRTArtificial SequenceSynthetic 112Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Val Asp Glu Cys Cys Glu 20 25 30 Pro Leu Glu Cys Phe Gln Met Gly His Gly Phe Lys Arg Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Asn 50 11348PRTArtificial SequenceSynthetic 113Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Phe Lys Arg Phe Ser Phe Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 11448PRTArtificial SequenceSynthetic 114Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Phe Lys Arg Phe Ser Phe Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Asn 35 40 45 11562PRTArtificial SequenceSynthetic 115Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Trp Ile Arg Phe Val Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Asp Cys Gly Thr Cys Met Phe Tyr Ser Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 11662PRTArtificial SequenceSynthetic 116Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Val Trp Phe Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Thr Tyr Gln Ser Gly Tyr Tyr Trp Leu Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 11762PRTArtificial SequenceSynthetic 117Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Leu Thr Gln Thr Arg Ser Cys Cys Glu 20 25 30 Pro Leu Glu Cys Arg Phe Leu Arg Ser His Ala Arg Thr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 11862PRTArtificial SequenceSynthetic 118Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Arg Lys Arg Tyr Arg Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Ile Leu Gln Phe Met Asn Lys Met Phe Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 11962PRTArtificial SequenceSynthetic 119Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Trp 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Pro Trp Tyr Trp Arg Cys Cys Glu 20 25 30 Pro Leu Glu Cys Val Tyr Thr Ser Gly Tyr Tyr Tyr Ser Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 12062PRTArtificial SequenceSynthetic 120Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Gly Ser Val Gly His Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg Arg Phe Leu Asn Leu Arg Trp Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 12162PRTArtificial SequenceSynthetic 121Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Ser Arg Ile Trp Ala Cys Cys Gly 20 25 30 Pro Leu Glu Cys Leu Met Arg Phe Met Ala Lys Arg Phe Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 12252PRTArtificial SequenceSynthetic 122Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Ala Lys Lys Asp Glu Leu Cys Ser Pro Ala Arg Arg Ile Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg Arg Trp Tyr Glu Glu Ser Phe Cys Gly Ser 35 40 45 Gly Ser Ser Gly 50 12362PRTArtificial SequenceSynthetic 123Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Thr Met Asn Glu Val Cys Cys Cys Glu 20 25 30 Pro Leu Glu Cys Tyr Gly Asp Ile Ser Gly Glu Ala Met Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 12462PRTArtificial SequenceSynthetic 124Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ile Val Met Arg Ala Pro Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg Arg Tyr Met Leu Lys His Met Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 12562PRTArtificial SequenceSynthetic 125Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Lys Ala Phe Arg Tyr Ser Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg Lys Trp Leu Lys Ala Arg Phe Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 12655PRTArtificial SequenceSynthetic 126Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Val Ser Gly Leu Met Asn Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Trp Arg Trp Met Gln Lys Gln Gln Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser His 50 55 12755PRTArtificial SequenceSynthetic 127Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Trp Arg Pro Ala Ile Thr Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg Ile Tyr Met Arg Leu Trp Arg Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu 50 55 12856PRTArtificial SequenceSynthetic 128Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ser Gln Leu Asp Ser Ala Cys Cys Glu 20 25 30 Pro Leu Glu Cys Val Trp Gln Asn Asp Asn Tyr Gly Thr Cys Gly Arg 35 40 45 Ala Val Leu Gly Leu Ala His Pro 50 55 12950PRTArtificial SequenceSynthetic 129Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Met Ser Met Val Gln Ile Cys Cys Glu 20 25 30 Pro Leu Glu Cys Phe His Ile Val Trp Cys Pro Trp Ala Cys Thr Ala 35 40 45 Val His 50 13051PRTArtificial SequenceSynthetic 130Met His His His His His His Ser Asp Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Leu Met Arg Val Leu Arg Cys Cys Glu 20 25 30 Pro Leu Glu Cys Trp Val Gly Gly Val Cys Arg Gly Gly Cys Gly Ser 35 40 45 Gly Ser Tyr 50 13162PRTArtificial SequenceSynthetic 131Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Thr Lys Ile Phe Lys Arg Cys Cys Glu 20 25 30 Pro Leu Glu Cys Ser Trp Val Val Trp Phe Pro Tyr Ser Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 13262PRTArtificial SequenceSynthetic 132Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Lys Lys Ile Asn Ala Lys Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg Arg Phe Leu Arg Phe Lys Phe Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 13362PRTArtificial SequenceSynthetic 133Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Leu Arg Ser Ser Ile Asp Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Tyr Lys Trp Met Gln Arg Arg Leu Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 13462PRTArtificial SequenceSynthetic 134Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Gly Leu Leu Thr Ser Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Val Trp Val Leu His His Phe Val Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 13558PRTArtificial SequenceSynthetic 135Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Thr Thr Ala Ser Lys Ser Cys Cys Glu 20 25 30 Pro Leu Glu Cys Lys Trp Thr Asn Glu His Phe Gly Thr

Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Tyr 50 55 13652PRTArtificial SequenceSynthetic 136Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Ile Tyr Thr Ser Glu Leu Tyr Ala Thr Cys Gly Ser 35 40 45 Gly Ser Ser Gly 50 13760PRTArtificial SequenceSynthetic 137Met His His His His His His Ser Asp Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Arg Asn Val Arg Thr Pro Cys Cys Glu 20 25 30 Pro Leu Glu Cys Asp Leu Phe Leu Thr Phe Leu Phe Leu Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Asn 50 55 60 13857PRTArtificial SequenceSynthetic 138Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Met Ser Met Ala Cys Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Lys Tyr His Gly Tyr Phe Trp Leu Cys Gly Ser Ala 35 40 45 Val Leu Gly Pro Ser Ser Ile Arg Tyr 50 55 13956PRTArtificial SequenceSynthetic 139Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Phe Trp Trp Leu Thr Leu Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg His Ile Cys Leu Val Ser Pro Cys Gly Arg 35 40 45 Ala Val Leu Gly Leu Ala His Pro 50 55 14062PRTArtificial SequenceSynthetic 140Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Arg Lys Arg Arg Asn Gly His Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Trp Trp Ala Gly Val Pro Leu Met Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 14162PRTArtificial SequenceSynthetic 141Met His His His His His His Ser Asp Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Val Trp Asn Ser Met Pro Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg His Arg Leu Leu Leu Arg Leu Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 14262PRTArtificial SequenceSynthetic 142Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Arg Pro Glu Val Leu Ser Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Arg Arg Trp Phe Gln Lys Arg Met Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 14362PRTArtificial SequenceSynthetic 143Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Phe Ser Arg Met Phe Met Cys Cys Glu 20 25 30 Pro Leu Glu Cys Asn Cys Pro Leu Ile Met Phe Ile Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 14462PRTArtificial SequenceSynthetic 144Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Trp Pro Arg Arg His Lys Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Leu Glu Met Leu Glu Arg Lys Arg Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 14562PRTArtificial SequenceSynthetic 145Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys His Ala Trp Tyr Thr Phe Cys Cys Glu 20 25 30 Pro Leu Glu Cys Gln Arg Lys Phe Gly Gly Tyr Trp Ala Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 14662PRTArtificial SequenceSynthetic 146Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Trp Glu Asp Met Thr Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Pro Ala Leu Glu Ser Val Val Leu Gln Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55 60 14750PRTArtificial SequenceSynthetic 147Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Leu Cys Trp Gln Trp Thr Cys Cys Glu 20 25 30 Pro Leu Glu Cys Glu Leu Gln Trp Gly Ile Ile Arg Met Cys Gly Ser 35 40 45 Gly Asn 50 14860PRTArtificial SequenceSynthetic 148Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 14958PRTArtificial SequenceSynthetic 149Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Arg 35 40 45 Ala Val Leu Gly Leu Ala His Pro Leu Phe 50 55 15060PRTArtificial SequenceSynthetic 150Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Phe Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 15159PRTArtificial SequenceSynthetic 151Met His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys Val 1 5 10 15 Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu Pro 20 25 30 Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser Gly 35 40 45 Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 15260PRTArtificial SequenceSynthetic 152Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Asp Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 15350PRTArtificial SequenceSynthetic 153Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Asn 50 15460PRTArtificial SequenceSynthetic 154Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Gly 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 15547PRTArtificial SequenceSynthetic 155Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35 40 45 15646PRTArtificial SequenceSynthetic 156Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys 35 40 45 15748PRTArtificial SequenceSynthetic 157Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Arg 35 40 45 15861PRTArtificial SequenceSynthetic 158Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Phe Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Asn 50 55 60 15960PRTArtificial SequenceSynthetic 159Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Phe Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 16060PRTArtificial SequenceSynthetic 160Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Trp Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 16160PRTArtificial SequenceSynthetic 161Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Val Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 16255PRTArtificial SequenceSynthetic 162Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser His 50 55 16359PRTArtificial SequenceSynthetic 163Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Arg Ser Pro Thr Leu Ser Cys Cys Glu Pro Leu Glu 20 25 30 Cys Leu Arg Val Tyr Leu Glu His Trp Phe Cys Gly Ser Gly Ser Gly 35 40 45 Ser Ser Leu Val Ala Ser Ala Ser Ala Ile Asn 50 55 16460PRTArtificial SequenceSynthetic 164Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Gln Leu Cys Ala Leu His Cys Cys Glu 20 25 30 Pro Leu Glu Cys Val Arg Met Met Phe Leu Val His Arg Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 16561PRTArtificial SequenceSynthetic 165Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Asn Trp Val Thr Pro Met Arg Cys Glu 20 25 30 Pro Leu Glu Cys Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35 40 45 Ser Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 16661PRTArtificial SequenceSynthetic 166Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Val Phe Tyr Phe Pro Asn Cys Cys Glu 20 25 30 Pro Leu Glu Cys Val Arg Trp Val Asn Asp Asn Tyr Gly Trp Cys Gly 35 40 45 Ser Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 16761PRTArtificial SequenceSynthetic 167Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Thr Thr Ala Ser Lys Ser Cys Cys Glu 20 25 30 Pro Leu Glu Cys Val Lys Trp Thr Asn Glu His Phe Gly Thr Cys Gly 35 40 45 Ser Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 16858PRTArtificial SequenceSynthetic 168Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Met Ser Met Ala Cys Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Val Lys Tyr His Gly Tyr Phe Trp Leu Cys Cys Gly 35 40 45 Ser Gly Ser Ser Gly Ser Ser Leu Val Glu 50 55 16961PRTArtificial SequenceSynthetic 169Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ser Val Trp Tyr Arg Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Val Thr Pro Asp Trp Ser Gly Ile Leu Tyr Cys Gly 35 40 45 Ser Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 17052PRTArtificial SequenceSynthetic 170Ser Ser Ser Gly Ser Gly Cys Val Lys Lys Asp Glu Leu Cys Glu Leu 1 5 10 15 Ala Ile Asp Val Cys Cys Glu Pro Leu Glu Cys Val Leu Gly His Gly 20 25 30 Leu Gly Tyr Ala Tyr Cys Gly Ser Gly Ser Ser Gly Ser Ser Leu Val 35 40 45 Ala Ser Ala Ile 50 17160PRTArtificial SequenceSynthetic 171Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50

55 60 17260PRTArtificial SequenceSynthetic 172Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Val Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 17358PRTArtificial SequenceSynthetic 173Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ile Leu Gly Leu Ala His Pro Leu Phe 50 55 17460PRTArtificial SequenceSynthetic 174Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Val Phe 50 55 60 17556PRTArtificial SequenceSynthetic 175Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Leu Gly Leu Ala His Pro 50 55 17654PRTArtificial SequenceSynthetic 176Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Arg 50 17753PRTArtificial SequenceSynthetic 177Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Leu 50 17855PRTArtificial SequenceSynthetic 178Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser His 50 55 17954PRTArtificial SequenceSynthetic 179Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser 50 18054PRTArtificial SequenceSynthetic 180Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Arg 50 18145PRTArtificial SequenceSynthetic 181Ser Gly Ser Ser Ser Gly Ser Gly Cys Val Lys Lys Asp Glu Leu Cys 1 5 10 15 Glu Leu Ala Ile Asp Val Cys Cys Glu Pro Leu Glu Cys Leu Gly His 20 25 30 Gly Leu Gly Tyr Ala Tyr Cys Gly Ser Gly Ser Ser Gly 35 40 45 18252PRTArtificial SequenceSynthetic 182Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Asp 50 18351PRTArtificial SequenceSynthetic 183Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser 50 18451PRTArtificial SequenceSynthetic 184Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Tyr 50 18551PRTArtificial SequenceSynthetic 185Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Cys Ser Tyr 50 18650PRTArtificial SequenceSynthetic 186Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Arg 50 18749PRTArtificial SequenceSynthetic 187Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Thr 35 40 45 Ala 18849PRTArtificial SequenceSynthetic 188Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly 18945PRTArtificial SequenceSynthetic 189Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr 35 40 45 19060PRTArtificial SequenceSynthetic 190Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Val Phe 50 55 60 19151PRTArtificial SequenceSynthetic 191Met His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys Val Lys Lys 1 5 10 15 Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu Pro Leu Glu 20 25 30 Cys Trp Leu Gly His Gly Leu Gly Tyr Ala His Cys Gly Ser Gly Ser 35 40 45 Ser Gly Ser 50 19261PRTArtificial SequenceSynthetic 192Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Glu Cys Cys Glu 20 25 30 Pro Leu Glu Cys Val Thr Lys Gly Asp Leu Gly Phe Arg Lys Cys Gly 35 40 45 Ser Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 19350PRTArtificial SequenceSynthetic 193Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser 50 19450PRTArtificial SequenceSynthetic 194Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Tyr 50 19554PRTArtificial SequenceSynthetic 195Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Ile Tyr Thr Ser Glu Leu Tyr Ala Thr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Arg 50 19660PRTArtificial SequenceSynthetic 196Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Thr Thr Ala Ser Lys Ser Cys Cys Glu 20 25 30 Pro Leu Glu Cys Lys Trp Thr Asn Glu His Phe Gly Thr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 19758PRTArtificial SequenceSynthetic 197Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Thr Thr Ala Ser Lys Ser Cys Cys Glu 20 25 30 Pro Leu Glu Cys Lys Trp Thr Asn Glu His Phe Gly Thr Cys Gly Thr 35 40 45 Ala Val Leu Gly Leu Ala His Pro Leu Phe 50 55 19860PRTArtificial SequenceSynthetic 198Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Ser Gln Ser Val Pro Met Cys Cys Glu 20 25 30 Pro Leu Glu Cys Lys Trp Phe Asn Glu Asn Tyr Gly Ile Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 19960PRTArtificial SequenceSynthetic 199Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 20060PRTArtificial SequenceSynthetic 200Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Asp 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 20160PRTArtificial SequenceSynthetic 201Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Ala Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ile Arg Tyr 50 55 60 20259PRTArtificial SequenceSynthetic 202Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala 50 55 20358PRTArtificial SequenceSynthetic 203Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Ala Val Leu Gly Leu Ala His Pro Leu Phe 50 55 20457PRTArtificial SequenceSynthetic 204Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala 50 55 20555PRTArtificial SequenceSynthetic 205Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser His 50 55 20660PRTArtificial SequenceSynthetic 206Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Pro Ser Ser Ile Arg Tyr 50 55 60 20758PRTArtificial SequenceSynthetic 207Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Thr 35 40 45 Ala Val Leu Gly Leu Ala His Pro Leu Phe 50 55 20858PRTArtificial SequenceSynthetic 208Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Arg Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Leu Gly Leu Ala His Pro Leu Phe 50 55 20953PRTArtificial SequenceSynthetic 209Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser

35 40 45 Gly Ser Ser Gly Ser 50 21058PRTArtificial SequenceSynthetic 210Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Arg 35 40 45 Ala Val Leu Gly Leu Ala His Pro Leu Phe 50 55 21158PRTArtificial SequenceSynthetic 211Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser 50 55 21260PRTArtificial SequenceSynthetic 212Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Thr Thr Ala Ser Lys Ser Cys Cys Glu 20 25 30 Pro Leu Glu Cys Lys Trp Thr Asn Glu His Phe Gly Thr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 21361PRTArtificial SequenceSynthetic 213Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile Tyr 50 55 60 21451PRTArtificial SequenceSynthetic 214Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Tyr 50 21557PRTArtificial SequenceSynthetic 215Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Glu Xaa 50 55 21661PRTArtificial SequenceSynthetic 216Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Arg Tyr 50 55 60 21747PRTArtificial SequenceSynthetic 217Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35 40 45 21856PRTArtificial SequenceSynthetic 218Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Asp 50 55 21954PRTArtificial SequenceSynthetic 219Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Val Leu Gly Leu Ala 50 22059PRTArtificial SequenceSynthetic 220Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala 50 55 22161PRTArtificial SequenceSynthetic 221Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Glu Cys Cys Glu 20 25 30 Pro Leu Glu Cys Thr Lys Gly Asp Leu Gly Phe Arg Lys Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile Tyr 50 55 60 22259PRTArtificial SequenceSynthetic 222Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Glu Phe Trp Val Pro Ser Ser Ile Arg Tyr Leu 50 55 22358PRTArtificial SequenceSynthetic 223Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser 50 55 22453PRTArtificial SequenceSynthetic 224Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser 50 22555PRTArtificial SequenceSynthetic 225Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser His 50 55 22658PRTArtificial SequenceSynthetic 226Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Ala Val Leu Gly Leu Ala His Pro Leu Phe 50 55 22754PRTArtificial SequenceSynthetic 227Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ile Leu Gly Leu Ala 50 22860PRTArtificial SequenceSynthetic 228Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Asn 50 55 60 22957PRTArtificial SequenceSynthetic 229Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala 50 55 23052PRTArtificial SequenceSynthetic 230Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Asp 50 23160PRTArtificial SequenceSynthetic 231Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Phe 50 55 60 23256PRTArtificial SequenceSynthetic 232Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val 50 55 23358PRTArtificial SequenceSynthetic 233Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Leu Ala His Pro Leu Phe 50 55 23460PRTArtificial SequenceSynthetic 234Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Asn 50 55 60 23550PRTArtificial SequenceSynthetic 235Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Arg 50 23658PRTArtificial SequenceSynthetic 236Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Tyr 50 55 23759PRTArtificial SequenceSynthetic 237Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala 50 55 23860PRTArtificial SequenceSynthetic 238Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Asn 50 55 60 23959PRTArtificial SequenceSynthetic 239Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Gly Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala 50 55 24055PRTArtificial SequenceSynthetic 240Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu 50 55 24158PRTArtificial SequenceSynthetic 241Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Leu Gly Leu Ala His Pro Leu Tyr 50 55 24260PRTArtificial SequenceSynthetic 242Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 60 24360PRTArtificial SequenceSynthetic 243Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Arg Tyr 50 55 60 24456PRTArtificial SequenceSynthetic 244Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25 30 Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40 45 Gly Ser Ser Gly Ser Ser Leu Val 50 55 24560PRTArtificial SequenceSynthetic 245Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Met Trp Ser Arg Glu Val Cys Cys Glu 20 25 30 Leu Leu Glu Cys Tyr Tyr Thr Gly Trp Tyr Trp Ala Cys Gly Ser Gly 35 40 45 Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile Tyr 50 55 60 24658PRTArtificial SequenceSynthetic 246Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Ala Ala 1 5 10 15 Ser Arg Lys Thr Ser Ser Ala Ser Trp Arg Ser

Thr Cys Ala Val Ser 20 25 30 Pro Ser Ser Ala Trp Gly Thr Ala Trp Gly Thr Arg Thr Ala Ala Ala 35 40 45 Ala Val Leu Gly Leu Ala His Pro Leu Phe 50 55 24759PRTArtificial SequenceSynthetic 247Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Ala Ala 1 5 10 15 Ser Arg Lys Thr Ser Ser Ala Ser Trp Arg Ser Thr Cys Ala Val Ser 20 25 30 Pro Ser Ser Ala Trp Gly Thr Ala Trp Gly Thr Arg Thr Ala Ala Ala 35 40 45 Ala Val Leu Gly Leu Ala His Pro Pro Ile Tyr 50 55 24856PRTArtificial SequenceSynthetic 248Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Ala Ala 1 5 10 15 Ser Arg Lys Thr Ser Ser Ala Ser Trp Arg Ser Thr Cys Ala Val Ser 20 25 30 Pro Ser Ser Ala Trp Gly Thr Ala Trp Gly Thr Arg Thr Ala Ala Ala 35 40 45 Ala Val Leu Gly Leu Ala His His 50 55 24957PRTArtificial SequenceSynthetic 249Met His His His His His His Ser Gly Lys Leu Arg Ile Leu Arg Gln 1 5 10 15 Glu Arg Arg Ala Leu Arg Ala Gly Asp Arg Arg Val Leu Ala Pro Arg 20 25 30 Val Leu Gly Ala Arg Pro Gly Val Arg Val Leu Arg Gln Arg Gln Phe 35 40 45 Trp Val Pro Ser Ser Ile Arg Tyr Leu 50 55 25058PRTArtificial SequenceSynthetic 250Met His His His His His His Ser Gly Ser Thr Gln Asp Gln Ala Ala 1 5 10 15 Ser Arg Lys Thr Ser Ser Ala Ser Trp Arg Ser Thr Cys Ala Val Ser 20 25 30 Pro Ser Ser Ala Trp Gly Thr Ala Trp Gly Thr Arg Thr Ala Ala Ala 35 40 45 Ala Val Leu Gly Leu Ala His Pro Leu Phe 50 55 25154PRTArtificial SequenceSynthetic 251Met His His His His His Gln Val Ala Gln Leu Arg Ile Arg Leu Arg 1 5 10 15 Gln Glu Arg Arg Ala Leu Arg Ala Gly Asp Arg Arg Val Leu Ala Pro 20 25 30 Arg Val Leu Gly Ala Arg Pro Gly Val Arg Val Leu Arg Gln Arg Gln 35 40 45 Phe Trp Val Pro Ser Ser 50 25259PRTArtificial SequenceSynthetic 252Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys 1 5 10 15 Val Lys Lys Asp Glu Leu Cys Met Trp Ser Arg Glu Val Cys Cys Glu 20 25 30 Leu Leu Glu Cys Tyr Tyr Thr Gly Trp Tyr Trp Ala Cys Gly Ser Gly 35 40 45 Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55 25350PRTArtificial SequenceSynthetic 253Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Val 1 5 10 15 Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 25450PRTArtificial SequenceSynthetic 254Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 25550PRTArtificial SequenceSynthetic 255Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Arg 1 5 10 15 Asp Thr Arg Lys Leu His Met Arg His Tyr Phe Pro Leu Ala Ile Asp 20 25 30 Ser Tyr Trp Asp His Thr Leu Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 25650PRTArtificial SequenceSynthetic 256Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Thr 1 5 10 15 Ser Met Gln Gly Glu Thr Leu Trp Arg Thr Asp Arg Leu Ala Thr Thr 20 25 30 Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 25750PRTArtificial SequenceSynthetic 257Met Gly Arg Gly Ser His His His His His His His Ala Arg Ser Pro 1 5 10 15 Leu Trp Tyr His Tyr Asn Cys Trp Asp Thr Ile Cys Leu Ala Asp Trp 20 25 30 Leu Lys Asp Arg Pro His Gly Val Tyr Asp Ala Asn Ala Pro Lys Ala 35 40 45 Ser Ala 50 25850PRTArtificial SequenceSynthetic 258Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Cys Leu 1 5 10 15 Ala Thr Arg Asn Gly Phe Val Gln Met Asn Thr Asp Arg Gly Thr Tyr 20 25 30 Val Lys Arg Pro Thr Val Leu Gln Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 25950PRTArtificial SequenceSynthetic 259Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Ser 1 5 10 15 Ala Leu Arg Arg Thr Glu Arg Thr Trp Asn Thr Ile His Gln Gly His 20 25 30 His Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 26050PRTArtificial SequenceSynthetic 260Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Thr Met 1 5 10 15 Asn Thr Asn Arg Met Asp Ile Gln Arg Leu Met Thr Asn His Val Lys 20 25 30 Arg Asp Ser Ser Pro Gly Ser Ile Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 26150PRTArtificial SequenceSynthetic 261Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asp Trp 1 5 10 15 Glu Leu Ser Pro Pro His Val Ala Ile Thr Thr Arg His Leu Ile Asn 20 25 30 Cys Thr Asp Gly Pro Leu Leu Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 26250PRTArtificial SequenceSynthetic 262Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Asn 1 5 10 15 Gly Glu Ser Thr Ser Asn Ile Leu Thr Thr Ser Arg Lys Val Thr Glu 20 25 30 Trp Thr Gly Tyr Thr Ala Ser Val Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 26350PRTArtificial SequenceSynthetic 263Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Gln Val 1 5 10 15 Thr Trp His His Leu Ala Asp Thr Val Thr Thr Lys Asn Arg Lys Cys 20 25 30 Thr Asp Ser Tyr Ile Gly Trp Asn Xaa Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 26450PRTArtificial SequenceSynthetic 264Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ile Ile 1 5 10 15 Val Ile His Asn Ala Ile Gln Thr His Thr Pro His Gln Val Ser Ile 20 25 30 Trp Cys Pro Pro Lys His Asn Arg Asp Asp Ala Asn Ala Pro Lys Ala 35 40 45 Ser Ala 50 26550PRTArtificial SequenceSynthetic 265Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ser His 1 5 10 15 Cys Arg His Arg Asn Cys His Thr Ile Thr Arg Gly Asn Met Arg Ile 20 25 30 Glu Thr Pro Asn Asn Ile Arg Lys Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 26650PRTArtificial SequenceSynthetic 266Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Gly 1 5 10 15 Leu Ser Gly Thr Gln Thr Trp Lys Ile Thr Lys Leu Ala Thr Arg Leu 20 25 30 His His Pro Glu Phe Glu Thr Asn Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 26750PRTArtificial SequenceSynthetic 267Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Arg 1 5 10 15 Trp His Asn Trp Gly Leu Ser Asp Thr Val Ala Ser His Pro Asp Ala 20 25 30 Ser Asn Ser Leu Asn Met Met Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Asn 50 26849PRTArtificial SequenceSynthetic 268Met Gly Arg Gly Ser His His His His His His Leu Asp Leu Trp Gly 1 5 10 15 Pro Pro Ser Gly Ser Pro Arg Thr Arg Ser Thr Thr Gly Thr Ser Thr 20 25 30 Thr Ser Ser Pro Ser Thr Pro Gly Thr Leu Thr Leu Arg Arg His Pro 35 40 45 His 26949PRTArtificial SequenceSynthetic 269Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Gln 1 5 10 15 Pro Glu Val Lys Met Ser Ser Leu Val Asp Thr Ser Gln Thr Val Gly 20 25 30 Ala Ala Val Glu Thr Arg Thr Thr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 27050PRTArtificial SequenceSynthetic 270Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Thr 1 5 10 15 Ser Met Gln Gly Glu Thr Leu Trp Arg Thr Asp Arg Leu Ala Thr Thr 20 25 30 Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 27150PRTArtificial SequenceSynthetic 271Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Ser 1 5 10 15 Ala Leu Arg Arg Thr Glu Arg Thr Trp Asn Thr Ile His Gln Gly His 20 25 30 His Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 27250PRTArtificial SequenceSynthetic 272Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Ser 1 5 10 15 Ala Leu Arg Arg Thr Glu Arg Thr Trp Asn Thr Ile His Gln Gly His 20 25 30 His Leu Glu Trp Tyr Pro Thr Ala Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 27350PRTArtificial SequenceSynthetic 273Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Lys Asp 1 5 10 15 Thr Ala Arg Thr Thr Ala Thr Leu Leu Thr Asn Asp Glu Asp Arg Lys 20 25 30 Thr His Trp Arg Met Phe Tyr Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 27450PRTArtificial SequenceSynthetic 274Met Gly Arg Gly Ser His His His His Tyr His Ala Arg Ser Lys Asp 1 5 10 15 Thr Ala Arg Thr Thr Ala Thr Leu Leu Thr Asn Asp Glu Asp Arg Lys 20 25 30 Thr His Trp Arg Met Phe Tyr Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 27550PRTArtificial SequenceSynthetic 275Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Thr Pro 1 5 10 15 Arg Leu Arg Lys Val Tyr Asp Leu Thr Val Thr Thr Thr Ser Ser Gln 20 25 30 Ile Asp Lys Leu Gln Pro Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 27650PRTArtificial SequenceSynthetic 276Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ser His 1 5 10 15 Cys Arg His Arg Asn Cys His Thr Ile Thr Arg Gly Asn Met Arg Ile 20 25 30 Glu Thr Pro Asn Asn Ile Arg Lys Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 27750PRTArtificial SequenceSynthetic 277Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asp Trp 1 5 10 15 Glu Leu Ser Pro Pro His Val Ala Ile Thr Thr Arg His Leu Ile Asn 20 25 30 Cys Thr Asp Gly Pro Leu Leu Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 27850PRTArtificial SequenceSynthetic 278Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ile Ser 1 5 10 15 Leu Ala Gln Tyr Tyr Trp Thr Ala Gln Arg Asp Met His Leu Leu Ile 20 25 30 Met His Lys Phe Met Asp Met Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 27950PRTArtificial SequenceSynthetic 279Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ile Ile 1 5 10 15 Val Ile His Asn Ala Ile Gln Thr His Thr Pro His Gln Val Ser Ile 20 25 30 Trp Cys Pro Pro Lys His Asn Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 28050PRTArtificial SequenceSynthetic 280Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Lys Phe 1 5 10 15 Arg Gln Ile Trp Glu Asn Glu Arg Lys Ala His Arg Met Val Met His 20 25 30 Gln Phe Tyr Gln Val Ile Arg Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 28150PRTArtificial SequenceSynthetic 281Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Ile 1 5 10 15 Val Cys Val Cys Thr Thr Ala Gly Asn Tyr Asn His His Asp Gly Phe 20 25 30 Phe Lys Arg Tyr Asp Asn Ser Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 28250PRTArtificial SequenceSynthetic 282Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Asn 1 5 10 15 Gly Glu Ser Thr Ser Asn Ile Leu Thr Thr Ser Arg Lys Val Thr Glu 20 25 30 Trp Thr Gly Tyr Thr Ala Ser Val Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 28350PRTArtificial SequenceSynthetic 283Ile Gly Arg Gly Ser His His His His His His Ala Arg Ser Ser Tyr 1 5 10 15 Pro Asp His Gly Arg Tyr Arg Asn Gln Ile Glu Arg Gly Thr Ile Glu 20 25 30 Met Thr Tyr Ile Asp Thr His Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 28449PRTArtificial SequenceSynthetic 284Met Gly Arg Gly Ser His His His His His Ala Arg Ser Gly Ala Glu 1 5 10 15 Pro Gly Met Ser Gly Lys Pro Lys Val Thr Thr Trp His His Lys Arg 20 25 30 Tyr Arg Arg Phe Met Thr His Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 28546PRTArtificial SequenceSynthetic 285Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asp Ile 1 5 10 15 Asp Thr Ala Glu Val Asn Arg Trp Glu Ser Asn Leu Lys Ser Tyr Leu 20 25 30 Tyr Asn Met Thr Asp Ala Asn Ala Pro Lys Ala Ser Ala Ile 35 40 45 28650PRTArtificial SequenceSynthetic 286Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Leu 1 5 10 15 Thr Gly Gln Ser Leu Tyr Tyr Gln Phe Met Ser Arg Ala Phe Phe Thr 20 25 30 Leu Gln Lys Phe Thr Gln Asn Leu Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 28750PRTArtificial SequenceSynthetic 287Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Lys Ile 1 5 10 15 Ala Glu Tyr Trp Leu Thr Glu Arg Met

Met His Leu Arg Ala Met Met 20 25 30 Lys Leu Leu Asn Lys His Ala His Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 28850PRTArtificial SequenceSynthetic 288Met Gly Arg Gly Ser His His His His His His Ala Arg Ser His Ser 1 5 10 15 Ala Leu Met His Asp Lys Asp Ser Ser Thr Ser Thr Tyr Tyr Pro Gln 20 25 30 Tyr Ala Asn Ser Pro Ser Val Gly Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 28950PRTArtificial SequenceSynthetic 289Met Gly Arg Gly Ser His His His His His His Ala Arg Ser His Leu 1 5 10 15 Asp Pro Cys Ala Asp Leu Asn Val Thr Gln Gln Arg Thr Thr Arg Glu 20 25 30 Thr His Ser Asp Asn Glu Asn His Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 29050PRTArtificial SequenceSynthetic 290Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Pro Leu 1 5 10 15 Tyr Gln Gly Glu Thr Leu Asn Ala Tyr Ala Pro Gln Ser Met Val Lys 20 25 30 Ile Ser Lys Asp Tyr Val Leu His Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 29150PRTArtificial SequenceSynthetic 291Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Tyr Met 1 5 10 15 Ala Arg Trp His Pro Met Thr His Asn His Met Lys Glu Thr Leu Phe 20 25 30 Ala Ala Glu Pro His Val Cys Thr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 29250PRTArtificial SequenceSynthetic 292Met Gly Arg Gly Ser His His His His His His Ala Arg Pro Arg Phe 1 5 10 15 His Pro Pro Phe Leu Arg Asp Arg Ser Val Asn Arg Met Ile Met Asn 20 25 30 Glu His Arg Pro Arg Tyr Ser His Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 29350PRTArtificial SequenceSynthetic 293Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ser Pro 1 5 10 15 Arg Tyr Ala Tyr Cys Gly Ser Arg Trp Asn Gly Ser Arg Met His Asn 20 25 30 Asn Lys Phe Thr Pro Ser Thr Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 29449PRTArtificial SequenceSynthetic 294Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Met 1 5 10 15 Asn Gln Met Thr Asn Ala Leu Asn Leu Arg Arg Arg Ser Arg Thr Trp 20 25 30 Val Ala Thr Phe Arg Ser Glu Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 29550PRTArtificial SequenceSynthetic 295Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Met Asn 1 5 10 15 Gly Leu Asp Met Gly Ser Pro Ile Trp Tyr Asn Met Gln Leu Lys Leu 20 25 30 Ile Tyr Phe Ser Cys Asn Trp Asn Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 29650PRTArtificial SequenceSynthetic 296Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Arg Val 1 5 10 15 Arg Asp Pro Asp Ser Gly Arg Thr His Gln Ile Arg Ser His Leu Lys 20 25 30 His Tyr Ser Asn Phe Pro Val Ala Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 29750PRTArtificial SequenceSynthetic 297Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Gln Val 1 5 10 15 Thr Trp His His Leu Ala Asp Thr Val Thr Thr Lys Asn Arg Lys Cys 20 25 30 Thr Asp Ser Tyr Ile Gly Trp Asn Xaa Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 29848PRTArtificial SequenceSynthetic 298Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ile Leu 1 5 10 15 Asp Val Asn Asp Glu Lys Arg Pro Pro Gly Trp Tyr Arg Thr Asn Ile 20 25 30 Ile Asp Ser Pro Ser Gly Asp Ala Asn Ala Pro Lys Ala Ser Ala Ile 35 40 45 29950PRTArtificial SequenceSynthetic 299Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Arg Arg 1 5 10 15 Tyr Arg Asp Gly Ile Phe Arg Arg Met Arg Ser Asx Thr Asn Ala Arg 20 25 30 Gly Ala Arg His Ala Asp Leu Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 30050PRTArtificial SequenceSynthetic 300Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Lys Cys 1 5 10 15 His Val Arg Arg Lys Glu Ser Ala Ser Ser Lys Asn Arg His Asn His 20 25 30 Thr Trp His Asp Ser Asn Leu Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 30150PRTArtificial SequenceSynthetic 301Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Arg Thr 1 5 10 15 Leu Leu Ile Arg Leu Tyr Pro Pro Asp Arg Phe Gly Ser Ser Arg Gln 20 25 30 Met Ala Thr Arg Asp Ser Phe Thr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 30250PRTArtificial SequenceSynthetic 302Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ser Gly 1 5 10 15 Met Tyr Val Val Ser Lys Pro Ala Ser Asp Ser Trp Thr Thr Cys Ala 20 25 30 Pro Tyr Thr Tyr Gly Thr Met Val Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 30350PRTArtificial SequenceSynthetic 303Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Leu 1 5 10 15 Ser Thr Ile Arg Asx Met Asn Arg His Leu Thr Asp Arg Arg Leu Thr 20 25 30 Ala Phe Arg Asn Gln Val Val Phe Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 30450PRTArtificial SequenceSynthetic 304Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ile Asn 1 5 10 15 Ala Trp Trp Tyr His Ile Gln Ser His Leu His Gln Trp Arg Arg His 20 25 30 Arg Leu Tyr Thr Ala Asn Gln Trp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 30550PRTArtificial SequenceSynthetic 305Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Thr Met 1 5 10 15 Asn Thr Asn Arg Met Asp Ile Gln Arg Leu Met Thr Asn His Val Lys 20 25 30 Arg Asp Ser Ser Pro Gly Ser Ile Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 30650PRTArtificial SequenceSynthetic 306Met Gly Arg Gly Ser His His His His His His Ala Arg Pro Asn Val 1 5 10 15 Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 30750PRTArtificial SequenceSynthetic 307Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Val 1 5 10 15 Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 30850PRTArtificial SequenceSynthetic 308Met Gly Arg Gly Ser His His His His His Arg Ala Arg Ser Asn Val 1 5 10 15 Ile Pro Leu Ser Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Thr 35 40 45 Ala Ile 50 30950PRTArtificial SequenceSynthetic 309Ile Glu Arg Gly Ser Gln His His His His His Ala Arg Ser Asn Val 1 5 10 15 Ile Thr Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Thr 35 40 45 Ala Ile 50 31049PRTArtificial SequenceSynthetic 310Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Val 1 5 10 15 Ile Thr Leu Ser Glu Val Trp Asp Thr Gly Trp Asn Arg Pro Leu Arg 20 25 30 Gln Arg Cys Arg Ser Glu Thr Asp Asp Asn Ala Gln Lys Ala Asn Asp 35 40 45 Ile 31150PRTArtificial SequenceSynthetic 311Met Gly Arg Gly Ser His His His His His Arg Ala Arg Ser Asn Val 1 5 10 15 Ile Pro Leu Ser Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 31250PRTArtificial SequenceSynthetic 312Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 31350PRTArtificial SequenceSynthetic 313Met Gly Arg Gly Ser Tyr His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 31451PRTArtificial SequenceSynthetic 314Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Asn Ala Asn Ala Pro Lys Ala 35 40 45 Ser Ala Ile 50 31550PRTArtificial SequenceSynthetic 315Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asn Ala Asn Ala Pro Lys Ala Thr 35 40 45 Ala Ile 50 31650PRTArtificial SequenceSynthetic 316Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Cys Arg Asn Ala Asn Ala Pro Lys Ala Thr 35 40 45 Ala Ile 50 31750PRTArtificial SequenceSynthetic 317Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Thr 1 5 10 15 Ser Met Gln Gly Glu Thr Leu Trp Arg Thr Asp Arg Leu Ala Thr Thr 20 25 30 Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 31850PRTArtificial SequenceSynthetic 318Met Gly Arg Gly Ser His His His His His Leu Ala Arg Ser Trp Thr 1 5 10 15 Ser Met Gln Gly Glu Thr Leu Trp Arg Thr Asp Arg Leu Ala Thr Thr 20 25 30 Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 31951PRTArtificial SequenceSynthetic 319Met Gly Arg Gly Ser His His His His His His His Ala Arg Ser Trp 1 5 10 15 Thr Ser Met Gln Gly Glu Thr Leu Trp Arg Thr Asp Arg Leu Ala Ala 20 25 30 Thr Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro Lys Ala 35 40 45 Ser Ala Ile 50 32050PRTArtificial SequenceSynthetic 320Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Ser 1 5 10 15 Ala Leu Arg Arg Thr Glu Arg Thr Trp Asn Thr Ile His Gln Gly His 20 25 30 His Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 32149PRTArtificial SequenceSynthetic 321Met Gly Arg Gly Ser His His His His His Ala Arg Ser Leu Ser Ala 1 5 10 15 Leu Arg Arg Thr Glu Arg Thr Trp Asn Thr Ile His Gln Gly His His 20 25 30 Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 32250PRTArtificial SequenceSynthetic 322Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Cys Leu 1 5 10 15 Ala Thr Arg Asn Gly Phe Val Gln Met Asn Thr Asp Arg Gly Thr Tyr 20 25 30 Val Lys Arg Pro Tyr Val Leu Gln Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 32350PRTArtificial SequenceSynthetic 323Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Lys Val 1 5 10 15 Asn Pro Met Arg Glu Val Arg Cys Asn Ala Arg Cys Ile Arg Lys His 20 25 30 Arg Phe Arg Leu Xaa Ile Arg Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 32450PRTArtificial SequenceSynthetic 324Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Thr Met 1 5 10 15 Asn Thr Asn Arg Met Asp Ile Gln Arg Leu Met Thr Asn His Val Lys 20 25 30 Arg Asp Ser Ser Pro Gly Ser Ile Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 32550PRTArtificial SequenceSynthetic 325Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Met Leu 1 5 10 15 Leu Leu Asn Glu Thr Tyr Arg Arg Tyr Arg Ser Trp Asp Glu Tyr Arg 20 25 30 Asn Asp Ile Gly Ser Asn Leu Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 32650PRTArtificial SequenceSynthetic 326Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Gly His 1 5 10 15 Arg Glu Ser Asn Arg Val Asn Ser Asn Tyr Ala Asp Gln Leu His Ser 20 25 30 Thr Pro Ile Leu Asn Thr Trp Asn Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 32750PRTArtificial SequenceSynthetic 327Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ser Gly 1 5 10 15 Gln Ile Pro Tyr Lys Tyr Gly Asp Ala Ile Pro Ser Met Leu Thr His 20 25 30 Asn Ala Glu Asn Gln Pro His Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 32850PRTArtificial SequenceSynthetic 328Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Thr Pro 1 5 10 15 Arg Leu Arg Lys Val Tyr Asp Leu Thr Val Thr Thr Thr Ser Ser Gln 20 25 30 Ile Asp Lys Leu Gln Pro Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 32950PRTArtificial SequenceSynthetic 329Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Glu Gly 1 5

10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 33050PRTArtificial SequenceSynthetic 330Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Met Arg 1 5 10 15 Pro Ile Leu Val Val Lys Tyr Pro Pro Tyr Leu Gln Thr Leu Asp Asn 20 25 30 Lys Arg Asp Ile Arg Gln Met Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 33150PRTArtificial SequenceSynthetic 331Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Lys Asn 1 5 10 15 Asn Thr Lys His Tyr Thr Val Val Thr Trp Cys Tyr Leu Glu Arg Lys 20 25 30 Asn Gln Asn Leu Thr Ser His Thr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 33250PRTArtificial SequenceSynthetic 332Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ile Leu 1 5 10 15 Arg Ser Ala Ser Cys Ser Ala Leu Thr Asp His Lys Arg Val Ala Tyr 20 25 30 Ala Cys Thr His Thr Glu Tyr Lys Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 33350PRTArtificial SequenceSynthetic 333Met Gly Arg Asp Ser His His His His His His Ala Arg Ser Ile Ala 1 5 10 15 Asn Met Tyr Gln Leu Trp Ser Met Asn Arg Ser Asp His Asn Leu Val 20 25 30 Ile Lys Lys Gln Met Ser Leu Leu Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 33450PRTArtificial SequenceSynthetic 334Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Met Leu 1 5 10 15 Leu Leu Asn Glu Thr Tyr Arg Arg Tyr Arg Ser Trp Asn Glu Tyr Arg 20 25 30 Asn Asp Ile His Ser Asn Leu Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 33550PRTArtificial SequenceSynthetic 335Met Gly Arg Gly Ser His His His His His His Thr Arg Ser Glu Glu 1 5 10 15 Asn Arg Gln Trp Arg Asn Glu Gly Ser Thr Pro Phe Ser Ser Leu Ile 20 25 30 Ser Asp Met Ser Lys Pro Ile Val Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 33650PRTArtificial SequenceSynthetic 336Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Val 1 5 10 15 Thr Arg Leu Leu Arg Thr His Arg Glu Glu Lys Val Phe Glu Pro Ser 20 25 30 Pro Thr Gly Pro Ser Glu Lys His Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 33749PRTArtificial SequenceSynthetic 337Met Gly Arg Gly Ser His His His His His Ala Arg Ser Asp Met Asp 1 5 10 15 Leu Trp Asp Leu Pro Ala Leu Ala Pro Gln Ser Thr Thr Met Gln Met 20 25 30 His Ser Phe Thr His Met Lys Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 33850PRTArtificial SequenceSynthetic 338Met Arg Arg Gly Ser His His His His His His Ala Arg Ser Arg Arg 1 5 10 15 Val Thr Thr Glu Gly Gly Pro Lys Trp Ile Pro Gly His His Met Arg 20 25 30 Asp Asn Ile Pro Glu Ile Ala Asn Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 33950PRTArtificial SequenceSynthetic 339Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Gly 1 5 10 15 Leu Ser Gly Thr Gln Thr Trp Lys Ile Thr Lys Leu Ala Thr Arg Leu 20 25 30 His His Pro Glu Phe Glu Thr Asn Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 34049PRTArtificial SequenceSynthetic 340Met Gly Arg Gly Ser His His His His His Ala Arg Ser Thr Trp Asn 1 5 10 15 Gly Arg Pro Leu His His Leu Asp His Gln Trp Tyr Pro Asp Glu Ala 20 25 30 Arg Leu His Ala Ile His Asn Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 34150PRTArtificial SequenceSynthetic 341Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Thr Asn 1 5 10 15 Arg Gly Val Asn His Thr Gly Gln Met Arg Thr Met Pro Pro Ala Pro 20 25 30 Thr Val Glu Arg Ala Leu Asn Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 34245PRTArtificial SequenceSynthetic 342Thr Gly Arg Gly Ser His His His His His His Ala Arg Ser Pro Leu 1 5 10 15 Glu Leu Tyr Val Ile Thr Arg Asp Ala Arg Thr Asp Thr Gly Pro Ser 20 25 30 Ser Leu Arg Asp Ala Asn Ala Pro Lys Ala Ser Ala Ile 35 40 45 34350PRTArtificial SequenceSynthetic 343Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Val 1 5 10 15 Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Pro Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 34449PRTArtificial SequenceSynthetic 344Met Gly Arg Gly Ser His His His His His Ala Arg Ser Asn Val Ile 1 5 10 15 Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His Arg 20 25 30 Ser Ser Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 34550PRTArtificial SequenceSynthetic 345Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 34650PRTArtificial SequenceSynthetic 346Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asn Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 34749PRTArtificial SequenceSynthetic 347Met Gly Arg Gly Ser His His His His His Ala Arg Ser Val Gly Thr 1 5 10 15 Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr His 20 25 30 Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 34849PRTArtificial SequenceSynthetic 348Met Gly Arg Gly Ser His His His His His Ala Arg Ser Val Gly Thr 1 5 10 15 Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr His 20 25 30 Lys Leu Ser His Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 34950PRTArtificial SequenceSynthetic 349Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Thr 1 5 10 15 Ser Met Gln Gly Glu Thr Leu Trp Arg Thr Asp Arg Leu Ala Thr Thr 20 25 30 Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 35049PRTArtificial SequenceSynthetic 350Met Gly Arg Gly Ser His His His His His Ala Arg Ser Pro Leu Trp 1 5 10 15 Tyr His Tyr Asn Cys Trp Asp Thr Ile Cys Leu Ala Asp Trp Leu Lys 20 25 30 Asp Arg Pro His Gly Val Tyr Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 35150PRTArtificial SequenceSynthetic 351Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Ser 1 5 10 15 Ala Leu Met Arg Thr Glu Arg Thr Trp Asn Thr Ile His Gln Gly His 20 25 30 His Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 35250PRTArtificial SequenceSynthetic 352Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Ser 1 5 10 15 Ala Leu Arg Arg Thr Glu Arg Thr Trp Asn Thr Ile His Gln Gly His 20 25 30 His Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 35349PRTArtificial SequenceSynthetic 353Met Gly Arg Gly Ser His His His His His Ala Arg Ser Leu Ser Ala 1 5 10 15 Leu Arg Arg Thr Glu Arg Thr Trp Asn Thr Ile His Gln Gly His His 20 25 30 Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 35449PRTArtificial SequenceSynthetic 354Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Cys Leu 1 5 10 15 Ala Thr Arg Asn Gly Phe Val Met Asn Thr Asp Arg Gly Thr Tyr Val 20 25 30 Lys Arg Pro Thr Val Leu Gln Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 35550PRTArtificial SequenceSynthetic 355Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Cys Leu 1 5 10 15 Ala Thr Arg Asn Gly Phe Val Gln Met Asn Thr Asp Arg Gly Thr Tyr 20 25 30 Val Lys Arg Pro Thr Val Leu Gln Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 35650PRTArtificial SequenceSynthetic 356Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Thr Met 1 5 10 15 Asn Thr Asn Arg Met Asp Ile Gln Arg Leu Met Thr Asn His Val Lys 20 25 30 Arg Asp Ser Ser Pro Gly Ser Ile Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 35750PRTArtificial SequenceSynthetic 357Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ser Phe 1 5 10 15 Asn Lys Val Gly Arg Val Asp Ser Glu Phe Gly Thr Lys Ala Asn Ser 20 25 30 His Gln Ile Pro Ser Gly Glu Leu Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 35850PRTArtificial SequenceSynthetic 358Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ile Lys 1 5 10 15 Tyr Trp Met Ile Pro Ser Trp Asn Leu Tyr Pro Trp Leu Leu Met Tyr 20 25 30 Asp Thr Leu Ile His Pro Thr Met Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 35950PRTArtificial SequenceSynthetic 359Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Trp 1 5 10 15 Thr Arg Met Gln Ile Pro Thr Ser Trp Tyr Trp Tyr Thr Tyr Trp Ile 20 25 30 Asn His Leu Gln Lys His Asp Ile Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 36050PRTArtificial SequenceSynthetic 360Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Arg 1 5 10 15 Trp His Asn Trp Gly Leu Ser Asp Thr Val Ala Ser His Pro Asp Ala 20 25 30 Ser Asn Ser Leu Asn Met Met Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 36150PRTArtificial SequenceSynthetic 361Met Gly Arg Gly Ser His His His His His Asp Ala Arg Ser Ser His 1 5 10 15 Trp Ser Asn Ala Asp His Ile Gly Pro Ser Arg Cys Leu Gly Cys Thr 20 25 30 Met Thr Thr Leu Ile Arg Leu Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 36250PRTArtificial SequenceSynthetic 362Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Arg Ser 1 5 10 15 Ile Pro Val Arg Ile Gln Gly Asn Pro Gly Asn Ser His Tyr Arg Leu 20 25 30 Met Gly Ala Ser Met Val His Gly Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 36350PRTArtificial SequenceSynthetic 363Met Gly Arg Asp Ser His His His His His His Ala Arg Ser Ile Ala 1 5 10 15 Asn Met Tyr Gln Leu Trp Ser Met Asn Arg Ser Asp His Asn Leu Val 20 25 30 Ile Lys Lys Gln Met Ser Leu Leu Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 36448PRTArtificial SequenceSynthetic 364Met Gly Arg Ser His His His His His Ala Arg Ser Gly Lys Phe Arg 1 5 10 15 His Glu Ile Tyr Asn Met Glu Trp Pro Leu Ala Leu Glu Arg Tyr Trp 20 25 30 Asp Tyr His Gly Glu Pro Asp Ala Asn Ala Pro Lys Ala Ser Ala Ile 35 40 45 36550PRTArtificial SequenceSynthetic 365Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Glu 1 5 10 15 Thr Thr Thr Thr Ser Leu Met Asn Glu Glu Asp Ala Trp Asn Trp Thr 20 25 30 Ile Glu Lys Ser Arg His Ile Glu Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 36650PRTArtificial SequenceSynthetic 366Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ile Met 1 5 10 15 Tyr Met His Trp Gln Trp Ala Val Asn Arg Met Gly His Ala Thr Ala 20 25 30 Met Ser Thr Leu Ala Asn Ala Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 36749PRTArtificial SequenceSynthetic 367Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Asp 1 5 10 15 Ile Pro Leu Asn Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His Arg 20 25 30 Ser Arg Leu Thr Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 36849PRTArtificial SequenceSynthetic 368Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Val 1 5 10 15 Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 36949PRTArtificial SequenceSynthetic 369Met Gly Arg Gly Ser His His His His His Arg Ala Arg Ser Asn Val 1 5 10 15 Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 37049PRTArtificial SequenceSynthetic 370Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 37149PRTArtificial SequenceSynthetic 371Met Gly Arg Gly Ser His His His His His His Thr Arg Ser Val Gly 1 5 10

15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 37248PRTArtificial SequenceSynthetic 372Met Gly Arg Gly Ser His His His Gln His Asn Ala Arg Ser Val Ala 1 5 10 15 Thr Thr Ile Pro Asp Arg Pro Gly His Gly Thr Leu Pro Glu Arg Leu 20 25 30 Pro Gln Ala Leu Pro Glu Leu Pro Gly Arg Arg Ser Glu Gly Ile Arg 35 40 45 37349PRTArtificial SequenceSynthetic 373Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 37449PRTArtificial SequenceSynthetic 374Met Gly Arg Gly Ser His Tyr His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 37548PRTArtificial SequenceSynthetic 375Met Gly Arg Gly Ser His His His His His Ala Arg Ser Val Gly Thr 1 5 10 15 Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr His 20 25 30 Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 37649PRTArtificial SequenceSynthetic 376Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Thr 1 5 10 15 Ser Met Gln Gly Glu Thr Leu Trp Arg Thr Asp Arg Leu Ala Thr Thr 20 25 30 Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 37749PRTArtificial SequenceSynthetic 377Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Gln 1 5 10 15 Pro Glu Val Lys Met Ser Ser Leu Val Asp Thr Ser Gln Thr Val Gly 20 25 30 Ala Ala Val Glu Thr Arg Thr Thr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 37849PRTArtificial SequenceSynthetic 378Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Ser 1 5 10 15 Ala Leu Arg Arg Thr Glu Arg Thr Trp Asn Thr Ile His Gln Gly His 20 25 30 His Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 37949PRTArtificial SequenceSynthetic 379Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Cys Leu 1 5 10 15 Ala Thr Arg Asn Gly Phe Val Gln Met Asn Thr Asp Arg Gly Thr Tyr 20 25 30 Val Lys Arg Pro Thr Val Leu Gln Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 38049PRTArtificial SequenceSynthetic 380Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Thr Met 1 5 10 15 Asn Thr Asn Arg Met Asp Ile Gln Arg Leu Met Thr Asn His Val Lys 20 25 30 Arg Asp Ser Ser Pro Gly Ser Ile Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 38149PRTArtificial SequenceSynthetic 381Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Gln Val 1 5 10 15 Thr Trp His His Leu Ala Asp Thr Val Thr Thr Lys Asn Arg Lys Cys 20 25 30 Thr Asp Ser Tyr Ile Gly Trp Asn Glu Leu Thr Leu Arg Arg His Pro 35 40 45 Leu 38249PRTArtificial SequenceSynthetic 382Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Thr Gly 1 5 10 15 Gly Pro Thr Gly Thr Ser Ala Ser Ala Gly Pro Thr Ser Ala Thr Arg 20 25 30 Ser Pro Pro Gly Gly Pro Arg Arg Thr Leu Thr Leu Arg Arg His Pro 35 40 45 Leu 38343PRTArtificial SequenceSynthetic 383Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Gly Lys 1 5 10 15 Val Arg Gly His Thr Lys Glu Thr Pro Pro Thr Glu Phe Gly Leu Ser 20 25 30 Leu Met Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 38449PRTArtificial SequenceSynthetic 384Met Gly Arg Gly Ser His His His His His His Leu Asp Leu Trp Gly 1 5 10 15 Pro Pro Ser Gly Ser Pro Arg Thr Arg Ser Thr Thr Gly Thr Ser Thr 20 25 30 Thr Ser Ser Pro Ser Thr Pro Gly Thr Leu Thr Leu Arg Arg His Pro 35 40 45 His 38549PRTArtificial SequenceSynthetic 385Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Pro Thr 1 5 10 15 Met Arg Arg His Ile Arg Arg Ala Leu Tyr Pro Tyr Ser Thr Arg Arg 20 25 30 Ser Leu Leu Thr Ser Ala Pro Val Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 38649PRTArtificial SequenceSynthetic 386Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ser Val 1 5 10 15 His Trp Ser Tyr Cys Gly Ala Glu Val Lys Lys Asp Trp Tyr Gln His 20 25 30 Thr Ala Trp Thr Lys Asn His Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 38749PRTArtificial SequenceSynthetic 387Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Met 1 5 10 15 Asn Thr Arg Arg Met Asp Ile Arg Asn Leu Ile Thr Lys Arg Val Lys 20 25 30 Lys Asp Tyr Ser Pro Gly Ser Lys Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 38849PRTArtificial SequenceSynthetic 388Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Asp 1 5 10 15 Asp Thr Gly His Leu Leu His Thr Gly Arg Leu Met Arg Thr Pro Ser 20 25 30 Thr Asn Ser Trp His Thr Leu Asn Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 38949PRTArtificial SequenceSynthetic 389Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ser Leu 1 5 10 15 Asn Lys Val Gly Arg Val Asp Ser Glu Phe Gly Thr Lys Ala Asn Ser 20 25 30 His Gln Ile Pro Ser Gly Glu Leu Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 39049PRTArtificial SequenceSynthetic 390Met Gly Arg Gly Ser His His His His His His Ala Arg Ser His Ser 1 5 10 15 Arg His Glu Trp Thr Ser Thr Pro Arg Arg Arg Arg Ser Thr Gly Pro 20 25 30 Gly Ser Arg Trp Ala Ser Gly Thr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 39149PRTArtificial SequenceSynthetic 391Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Gly Arg 1 5 10 15 Tyr His Arg Asp Arg Trp Leu Ala Thr Met Arg Tyr Pro Asp Pro Ser 20 25 30 Gln Val Trp Ser Arg Tyr Val Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 39249PRTArtificial SequenceSynthetic 392Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Arg 1 5 10 15 Trp His Asn Trp Gly Leu Ser Asp Thr Val Ala Ser His Pro Asp Ala 20 25 30 Ser Asn Ser Leu Asn Met Met Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 39349PRTArtificial SequenceSynthetic 393Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Pro Leu 1 5 10 15 Trp Tyr His Tyr Asn Cys Trp Asp Thr Ile Cys Leu Ala Asp Trp Leu 20 25 30 Lys Asp Arg Pro His Gly Val Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala 39449PRTArtificial SequenceSynthetic 394Met Gly Arg Gly Ser His His His His His Ala Arg Ser Asn Val Ile 1 5 10 15 Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His Arg 20 25 30 Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 39548PRTArtificial SequenceSynthetic 395Met Gly Leu Leu His His His His His Ala Arg Ser Asn Val Ile Pro 1 5 10 15 Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His Arg Ser 20 25 30 Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser Ala Ile 35 40 45 39650PRTArtificial SequenceSynthetic 396Met Gly Arg Ser Ser His His His His His His Ala Arg Ser Asn Val 1 5 10 15 Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 39750PRTArtificial SequenceSynthetic 397Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Val 1 5 10 15 Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 39849PRTArtificial SequenceSynthetic 398Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Val 1 5 10 15 Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Asn 39950PRTArtificial SequenceSynthetic 399Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Val 1 5 10 15 Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25 30 Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 40049PRTArtificial SequenceSynthetic 400Met Gly Arg Ser His His His His His His Ala Arg Ser Asn Val Ile 1 5 10 15 Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His Arg 20 25 30 Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40 45 Ile 40145PRTArtificial SequenceSynthetic 401Met Gly Arg Ser His His His His His His Ala Arg Ser Asn Val Ile 1 5 10 15 Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His Arg 20 25 30 Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Arg 35 40 45 40250PRTArtificial SequenceSynthetic 402Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Thr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 40344PRTArtificial SequenceSynthetic 403Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Thr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala 35 40 40450PRTArtificial SequenceSynthetic 404Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly 1 5 10 15 Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Thr Arg Asn Val Tyr 20 25 30 His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 40550PRTArtificial SequenceSynthetic 405Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Thr 1 5 10 15 Ser Met Gln Gly Glu Thr Leu Trp Arg Thr Asp Arg Leu Ala Thr Thr 20 25 30 Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 40650PRTArtificial SequenceSynthetic 406Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Cys Leu 1 5 10 15 Ala Thr Arg Asn Gly Phe Glu Gln Met Asn Thr Asp Arg Gly Thr Tyr 20 25 30 Val Lys Arg Thr Thr Val Leu Gln Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 40750PRTArtificial SequenceSynthetic 407Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Arg 1 5 10 15 Asp Thr Arg Lys Leu His Met Arg His Tyr Phe Pro Leu Ala Ile Asp 20 25 30 Ser Tyr Trp Asp His Thr Leu Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 40850PRTArtificial SequenceSynthetic 408Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ser Pro 1 5 10 15 Leu Trp Tyr His Tyr Asn Cys Trp Asp Thr Ile Cys Leu Ala Asp Trp 20 25 30 Leu Lys Asp Arg Pro His Gly Val Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 40951PRTArtificial SequenceSynthetic 409Met Gly Arg Gly Ser His His His His His His His Ala Arg Ser Pro 1 5 10 15 Leu Trp Tyr His Tyr Asn Cys Trp Asp Thr Ile Cys Leu Ala Asp Trp 20 25 30 Leu Lys Asp Arg Pro His Gly Val Tyr Asp Ala Asn Ala Pro Lys Ala 35 40 45 Ser Ala Ile 50 41050PRTArtificial SequenceSynthetic 410Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Gly Arg 1 5 10 15 Tyr His Arg Asp Arg Trp Leu Ala Thr Met Arg Tyr Pro Asp Pro Ser 20 25 30 Gln Val Trp Ser Arg Tyr Val Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 41150PRTArtificial SequenceSynthetic 411Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Thr Met 1 5 10 15 Asn Thr Asn Arg Met Asp Ile Gln Arg Leu Met Thr Asn His Val Lys 20 25 30 Arg Asp Ser Ser Pro Gly Ser Ile Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 41250PRTArtificial SequenceSynthetic 412Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Leu 1 5 10 15 Tyr Ile Thr Gly Glu Phe Lys Arg Gln Thr Asp Asn Asn Gly Ser Glu 20 25 30 Leu Arg Arg Met Ser Arg Pro Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 41350PRTArtificial SequenceSynthetic 413Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Cys 1 5 10 15 Leu Ile Ser Leu Thr Ala Glu Glu Lys Ala

Leu Asn Arg Met Met Asn 20 25 30 Val Ser Val Pro Arg Val Met Thr Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 41450PRTArtificial SequenceSynthetic 414Met Gly Arg Asp Ser His His His His His His Ala Arg Ser Ile Ala 1 5 10 15 Asn Met Tyr Gln Leu Trp Ser Met Asn Arg Ser Asp His Asn Leu Val 20 25 30 Ile Lys Lys Gln Met Ser Leu Leu Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50 41550PRTArtificial SequenceSynthetic 415Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Ser 1 5 10 15 Arg Leu Ala Thr Val Leu Asp Glu Pro Asp Arg Ser Leu Gln Thr Arg 20 25 30 Thr Asn Arg Pro His Arg Met Ile Asp Ala Asn Ala Pro Lys Ala Ser 35 40 45 Ala Ile 50

Claims

1-51. (canceled)

52. A method for isolating pertussis toxin comprising contacting pertussis toxin with an immobilized peptide comprising an amino acid sequence selected from the group consisting of: TABLE-US-00018 (SEQ ID NO.: 67) RSSHCRHRNCHTITRGNMRIETPNNIRKDAK; (SEQ ID NO.: 68) RSSHCRHRNCHTITRGNMRIETPNNIRKDA; (SEQ ID NO.: 69) RSTMNTNRMDIQRLMTNHVKRDSSPGSIDA; (SEQ ID NO.: 70) RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA; (SEQ ID NO.: 71) RSWRDTRKLHMRHYFPLAIDSYWDHTLRDA; (SEQ ID NO.: 72) SGCVKKDELCARWDLVCCEPLECIYTSELYATCGK; (SEQ ID NO.: 73) SGCVKKDELCARWDLVCCEPLECIYTSELYATCG; (SEQ ID NO.: 74) SGCVKKDELCELAVDECCEPLECFQMGHGFKRCG; (SEQ ID NO.: 75) SGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS; (SEQ ID NO.: 76) SGCVKKDELCELAIDECCEPLECTKGDLGFRKCG; and, (SEQ ID NO 14) CVKKDELCXXXXXXCCEPLECXXXXXXXXXC, where X is any amino acid.

53. The method of claim 52 wherein the immobilized peptide comprises the amino acid sequence RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA (SEQ ID NO.: 70).

54. The method of claim 52 wherein the pertussis toxin is isolated from a fermentation supernatant.

55. The method of claim 52 wherein the peptide is immobilized by binding to a solid support.

56. The method of claim 55 wherein the solid support is selected from selected from the group consisting of a column, chromatographic media, column material, bead, test tube, microtiter dish, solid particle, sepharose, agarose, microchip, silicon, silicon-glass, gold, and a membrane.

57. The method of claim 52 wherein the pertussis toxin is eluted from the peptide using a buffer comprising glycine, carbonate or magnesium.

58. The method of claim 57 wherein the buffer comprises glycine at about pH 2.5.

59. The method of claim 57 wherein the buffer comprises carbonate at about pH 10.5.

60. The method of claim 57 wherein the buffer comprises MgCl.sub.2.

61. The method of claim 56 wherein the solid support is a sepharose bead comprising 300-400 pmol of the peptide.

62. The method of claim 52 comprising a complex of a peptide and a solid support.

63. The method of claim 62 wherein the pertussis toxin is immobilized to a complex of a peptide and a solid support, the method further comprising eluting the pertussis toxin from the complex and regenerating the complex.

64. The method of claim 63 wherein the complex is regenerated using HCl.

65. The method of claim 52 wherein the immobilized peptide comprises the amino acid sequence RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA (SEQ ID NO.: 70) and the pertussis toxin is eluted from the peptide using a buffer comprising glycine at about pH 2.5 or carbonate at about pH 10.5.

Images