Patent application title: Methods for purifying pertussis toxin and peptides useful therefor
Inventors:
Andreas Jungbluth (Eppelborn-Humes, DE)
Eberhard Schneider (Denkte, DE)
Peter Wagner (Braunschweig, DE)
IPC8 Class: AC12P2100FI
USPC Class:
435 681
Class name: Chemistry: molecular biology and microbiology micro-organism, tissue cell culture or enzyme using process to synthesize a desired chemical compound or composition enzymatic production of a protein or polypeptide (e.g., enzymatic hydrolysis, etc.)
Publication date: 2009-08-27
Patent application number: 20090215113
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Patent application title: Methods for purifying pertussis toxin and peptides useful therefor
Inventors:
Andreas Jungbluth
Eberhard Schneider
Peter Wagner
Agents:
Patrick J. Halloran
Assignees:
Origin: CENTER VALLEY, PA US
IPC8 Class: AC12P2100FI
USPC Class:
435 681
Abstract:
The present invention relates to reagents and methods for purifying
pertussis toxin (PT).Claims:
1-51. (canceled)
52. A peptide having the ability to bind pertussis toxin, the peptide being selected from the group consisting of: TABLE-US-00018 RSSHCRHRNCHTITRGNMRIETPNNIRKDA (pp26-5); RSTMNTNRMDIQRLMTNHVKRDSSPGSIDA (pp26-6); RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA (pp26-9); RSWRDTRKLHMRHYFPLAIDSYWDHTLRDA (pp26-15); SGCVKKDELCARWDLVCCEPLECIYTSELYATCG (G-9); SGCVKKDELCELAVDECCEPLECFQMGHGFKRCG (G-10); SGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS (G-15); SGCVKKDELCELAIDECCEPLECTKGDLGFRKCG (G-19); NVIPLNEVWYDTGWDRPHRSRLSIDDD, VGTTIRIAQDTEHYRNVYHKLSQYSR, WTSMQGETLWRTDRLATTKTSMSHPP, LSALRRTERTWNTIHQGHHLEWYPPA, LSRLATTERTWDRIHQGHHLEWHPPA, TMNTNRMDIQRLMTNHVKRDSSPGSI, LSALMRTERTWNTIHQGHHLEWYPPA, CLATRNGFVMNTDRGTYVKRPTVLQ, and CLATRNGFVQMNTDRGTYVKRPTVLQ.
53. A peptide having the ability to bind pertussis toxin and the amino acid sequence RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA (pp26-9).
54. A peptide having the ability to bind pertussis toxin and the amino acid sequence SGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS (G-15).
55. A peptide of claim 52 wherein at least one amino acid is conservatively substituted.
56. A peptide of claim 53 wherein at least one amino acid is conservatively substituted.
57. A peptide of claim 54 wherein at least one amino acid is conservatively substituted.
58. A peptide having the ability to bind pertussis toxin and comprising the amino acid sequence selected from the group consisting of:XXAXRXXXXXXNTXXXXXXXXXT,XXAXRXXXXXXNTXXXXXXXXXY, andVXXXXXXXXDTXXXXRXXXXXLS,where X is any amino acid.
59. A peptide having the ability to bind pertussis toxin and comprising the amino acid sequence LGHGLGYAY.
60. A peptide of claim 59 further comprising the amino acid sequence ELAVD, ELAID, or ARWDLV.
61. A peptide having the ability to bind pertussis toxin and comprising at least one of the amino acid sequences TTASKS or KWTNEHFGT.
62. A peptide of claim 61 comprising the amino acid sequences TTASKS and KWTNEHFGT.
63. A method for generating a DNA-peptide fusion, said method comprising:(a) covalently bonding a nucleic acid reverse-transcription primer to an RNA encoding a peptide, said reverse-transcription primer being bound to a peptide acceptor;(b) translating said RNA to produce the peptide, the peptide being covalently bound to the reverse-transcription primer; and, (c) reverse transcribing said RNA to produce a DNA-peptide fusion;wherein the peptide of the DNA-peptide fusion has binding affinity for pertussis toxin.
64. A method for generating a DNA-peptide fusion, said method comprising:(a) generating an RNA-peptide fusion;(b) hybridizing a nucleic acid reverse-transcription primer to said fusion;(c) covalently bonding said primer to said fusion; and(d) reverse transcribing the RNA of said RNA-peptide fusion to produce a DNA-peptide fusion;wherein the peptide of the DNA-peptide fusion has binding affinity for pertussis toxin.
65. A method for generating a DNA-peptide fusion comprising:(a) providing an RNA molecule covalently bonded to a peptide acceptor;(b) covalently bonding a nucleic acid reverse-transcription primer to the molecule of step (a);(c) translating said RNA molecule to produce a peptide, and(d) reverse transcribing said RNA molecule to produce a DNA-peptide fusion;wherein the peptide of the DNA-peptide fusion has binding affinity for pertussis toxin.
66. A method for isolating a DNA-peptide fusion in which the peptide has binding affinity for pertussis toxin comprising the steps of, in combination:(a) covalently bonding a nucleic acid reverse-transcription primer to an RNA encoding a peptide, said reverse-transcription primer being bound to a peptide acceptor;(b) translating the RNA to produce the peptide, the peptide being covalently bound to the reverse-transcription primer; and,(c) reverse transcribing the RNA to produce a DNA-peptide fusion;(d) contacting the DNA-peptide fusion with pertussis toxin bound to a solid support to form a DNA-peptide fusion-pertussis toxin complex;(e) isolating the complex from DNA-peptide fusions that did not complex with pertussis toxin; and,(f) isolating the DNA-peptide fusion from the DNA-peptide fusion-pertussis toxin complex.
67. A method for purifying pertussis toxin comprising contacting a biological solution containing pertussis toxin with a peptide of claim 52 bound to a solid support to form a pertussis toxin-peptide complex and isolating the complex from other components in the biological solution.
68. A method for purifying pertussis toxin comprising contacting a biological solution containing pertussis toxin with a peptide of claim 53 bound to a solid support to form a pertussis toxin-peptide complex and isolating the complex from other components in the biological solution.
69. A method for purifying pertussis toxin comprising contacting a biological solution containing pertussis toxin with a peptide of claim 54 bound to a solid support to form a pertussis toxin-peptide complex and isolating the complex from other components in the biological solution.
70. A method for purifying pertussis toxin comprising contacting a biological solution containing pertussis toxin with a peptide of claim 55 bound to a solid support to form a pertussis toxin-peptide complex and isolating the complex from other components in the biological solution.
71. A method for purifying pertussis toxin comprising contacting a biological solution containing pertussis toxin with a peptide of claim 56 bound to a solid support to form a pertussis toxin-peptide complex and isolating the complex from other components in the biological solution.
72. A method for purifying pertussis toxin comprising contacting a biological solution containing pertussis toxin with a peptide of claim 57 bound to a solid support to form a pertussis toxin-peptide complex and isolating the complex from other components in the biological solution.
73. A method for purifying pertussis toxin comprising contacting a biological solution containing pertussis toxin with a peptide of claim 58 bound to a solid support to form a pertussis toxin-peptide complex and isolating the complex from other components in the biological solution.
74. A method for purifying pertussis toxin comprising contacting a biological solution containing pertussis toxin with a peptide of claim 59 bound to a solid support to form a pertussis toxin-peptide complex and isolating the complex from other components in the biological solution.
75. A method for purifying pertussis toxin comprising contacting a biological solution containing pertussis toxin with a peptide of claim 60 bound to a solid support to form a pertussis toxin-peptide complex and isolating the complex from other components in the biological solution.
76. A method for purifying pertussis toxin comprising contacting a biological solution containing pertussis toxin with a peptide of claim 61 bound to a solid support to form a pertussis toxin-peptide complex and isolating the complex from other components in the biological solution.
77. A method for purifying pertussis toxin comprising contacting a biological solution containing pertussis toxin with a peptide of claim 62 bound to a solid support to form a pertussis toxin-peptide complex and isolating the complex from other components in the biological solution.
78. A DNA-peptide fusion prepared using the method of claim 63.
79. A DNA-peptide fusion prepared using the method of claim 64.
80. A DNA-peptide fusion prepared using the method of claim 65.
81. A DNA-peptide fusion prepared using the method of claim 66.
Description:
FIELD OF THE INVENTION
[0001]The present invention relates to reagents and methods for purifying pertussis toxin (PT).
BACKGROUND OF THE INVENTION
[0002]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).
[0003]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; 3G8: NGSFSGC; and, 3G2: DGSFSGF) having the consensus sequence XGSFSGX (X is any amino acid) were identified as having PT-binding capacity. 3G2 was also utilized in an affinity column to purify PT from a partially purified PT preparation.
[0004]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
[0005]FIG. 1. A) Schematic representation of the gurmarin library. Positions of the library that are translated to an amino acid sequence are highlighted. 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.
[0006]FIG. 2. Schematic representation of a PROfusion® selection cycle.
[0007]FIG. 3. Selected gurmarin variants that should be tested for binding activity towards PT. Conserved sequence motifs are highlighted by colored boxes.
[0008]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 and constant regions of the gurmarin scaffold are highlighted. The position of the randomized loops 1 and 2 are indicated.
[0009]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 and constant regions of the gurmarin scaffold are highlighted. The position of the randomized loops 1 and 2 are indicated.
[0010]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 and constant regions of the gurmarin scaffold are highlighted. The position of the randomized loops 1 and 2 are indicated.
[0011]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 and constant regions of the gurmarin scaffold are highlighted. The position of the randomized loops 1 and 2 are indicated.
[0012]FIG. 8. Sequence analysis of the gurmarin selection round 6b against PT (strep). The ammo acid sequence of individual variants is shown in the single letter amino acid code. Constant, flanking regions of the library and constant regions of the gurmarin scaffold are highlighted. The position of the randomized loops 1 and 2 are indicated.
[0013]FIG. 9. Selected PP26 variants that will be tested for binding activity towards PT. Conserved sequence motifs are highlighted.
[0014]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 highlighted.
[0015]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.
[0016]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.
[0017]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.
[0018]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 highlighted.
[0019]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.
[0020]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.
[0021]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.
[0022]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.
[0023]FIG. 19. Elution of PT from peptide streptavidin sepharose with 0.2 to 2.0 MMgCl2 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.
[0024]FIG. 20. Elution of PT from peptide streptavidin sepharose under acidic (50 mM glycine, pH 2.5) or basic (100 mM carbonat 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 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.
[0025]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).
[0026]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).
[0027]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.
[0028]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.
[0029]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.
[0030]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
[0031]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
[0032]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, detoxified PT (genetically or otherwise), natural or other PT variants, recombinant PT, PT fragments, or other versions of PT (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. Any form of PT is suitable for use in practicing the present invention as long as a reagent as described herein has the ability to bind the particular form of PT. Within this application, all cited references, patents, and patent applications are incorporated herein by reference.
[0033]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).
[0034]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).
[0035]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 Kd) 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., concentration of an ionic salt such as sodium chloride or magnesium chloride, for example), solvent concentration, presence or absence of a competitor reagent/free ligand/analogue, polar properties, among others as is known in the art.
[0036]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.
[0037]To purify PT, a composition containing PT may be μlaced 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 Kd 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.
[0038]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 chromatography (LPC), high pressure liquid chromatography (HPLC), or fast protein liquid chromatography (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).
[0039]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 RSSHCRHRNCHTITRGNMRIETPNNIRKDA (pp26-5); STMNTNRMDIQRLMTNHVKRDSSPGSIDA (pp26-6); RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA (pp26-9); RSWRDTRKLHMRHYEPLAIDSYWDHTLRDA (pp26-15); SGCVKKDELCARWDLVCCEPLECIYTSELYATCG (G-9); SGCVKKDELCELAVDECCEPLECFQMGHGFKRCG (G-10); SGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS (G-15); and, SGCVKKDELCELAIDECCEPLECTKGDLGFRKCG (G-19).
Of these, especially preferred peptides include:
TABLE-US-00002 RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA (pp26-9); and, SGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS (G-15).
[0040]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.
[0041]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
[0042]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 as determined by SDS-PAGE, for example. 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.
[0043]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.
[0044]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)
[0045]PT is a heterooligomeric protein complex with a MWr of 109 kD (consists of the 6 subunits S1, S2, S3, 2x 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. Gurmarin Library Selection
[0046]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.
[0047]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.
[0048]A specialized gurmarin-library was designed with 15 randomized amino acid positions, as shown below:
TABLE-US-00004 Wild-type gumarin: qqCVKKDELCIPYYLDCCEPLECKKVNWWDHKCig Gumarin core: CVKKDELCXXXXXXCCEPLECXXXXXXXXXC
Within the gurmarin 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
[0049]Three gel-purified oligos were used to construct the gurmarin library with two randomized loops. 1 nmole of gurmarin template (≈ca. 61014 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', 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' and 1 μM of the 3'-Primer 5'-TTT TAA ATA GCG GAT GCT ACT AGG CTA GAC CCA GAA CTG CCG CT-3' using Taq-polymerase and analyzed on a 2% agarose gel, which indicated a representative library had been constructed.
2. In Vitro Transcription
[0050]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
[0051]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). 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
[0052]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
[0053]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
[0054]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
[0055]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
[0056]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 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 represents a His6-tag for efficient affinity purification of PROfusion® library. ARS represents a second structural, flexible linker. DANAPK represents a third structural, flexible linker. ASAI represents an optimized linker for efficient coupling with puromycin-acceptor-molecule.
[0057]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
[0058]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', 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' and 1 μM of the 3'-Primer 5'-AA TTA AAT AGC GGA TGC CTT CGG AGC GTT AGC -3' using Taq-polymerase and confirmed by analysis on a 2% agarose gel.
2. In Vitro Transcription
[0059]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
[0060]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). 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
[0061]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
[0062]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
[0063]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
[0064]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
[0065]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
[0066]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
[0067]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
[0068]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 pg, 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
[0069]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)
[0070]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)
[0071]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 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
[0072]2.6 mg dry epoxy-activated beads (M-270, Dynal) (˜1.7-108 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 continues 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
[0073]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.
[0074]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 beds 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
[0075]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
[0076]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
[0077]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
[0078]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
[0079]The gurmarin PROfusion® library and PT 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 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.
[0080]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
[0081]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.
[0082]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.
[0083]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'. 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
[0084]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
[0085]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
[0086]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'. 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
[0087]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
[0088]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
[0089]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.
[0090]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). 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.
[0091]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.
[0092]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 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.
[0093]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.
[0094]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 1 194227 MHHHHHHSGSSSGSGCVKKDELCAGSVGHCCEPLECLRRFLNLRWCGSGSSGSS -- n.d. 2 194238 MHHHHHHSGSSSGSGCVKKDELCIVMRAPCCEPLECLRRYMLKHMCGSGSSGSS -- n.d. 3 194239 MHHHHHHSGSSSGSGCVKKDELCKAFRYSCCEPLECLRKWLKARFCGSGSSGSS -- n.d. 4 194251 MHHHHHHSGSSSGSGCVKKDELCLRSSIDCCEPLECLYKWMQRRLCGSGSSGSS -- n.d. 5 194210 MHHHHHHSGSSSGSGCVKKDELCWPRRHKCCEPLECLLEMLERKRCGSGSSGSS -- n.d. 6 194261 MHHHHHHSGSSSGSGCVKKDELCMSMACVCCEPLECKYHGYFWLCGSGSSGSS -- n.d. 7 194214 MHHHHHHSGSSSGSGCVKKDELCAVWFDVCCEPLECTYQSGYYWLCGSGSSGSS -- n.d. 8 194226 MHHHHHHSGSSSGSGCVKKDELCEPWYWRCCEPLECVYTSGYYYSCGSGSSGSS -- n.d. 9 194259 MHHHHHHSGSSSGSGCVKKDELCARWDLVCCEPLECIYTSELYATCGSGSSGSS 12 194297 MHHHHHHSGSSSGSGCVKKDELCVFYFPNCCEPLECRWVNDNYGWCGSGSSGSS -- 13 194330 MHHHHHHSGSSSGSGCVKKDELCMSMACVCCEPLECKYHGYFWLCGSGSSGSS -- 14 194479 MHHHHHHSGSSSGSGCVKKDELCTTASKSCCEPLECKWTNEHFGTCGSGSSGSS -- 15 194511 MHHHHHHSGSSSGSGCVKKDELCSQSVPMCCEPLECKWFNENYGICGSGSSGSS -- 16 194533 MHHHHHHSGSSSGSGCVKKDELCARWDLVCCEPLECIYTSELYATCGSGSSGSS -- 17 194486 MHHHHHHSGSSSGSGCVKKDELCARWDLVCCEPLECLGHGLGYAYCGSGSSGSS -- n.d. 18 194668 MHHHHHHSGSSSGSGCVKKDELCMWSREVCCEPLECYYTGWYWACGSGSSGSS -- -- 10 194264 MHHHHHHSGSSSGSGCVKKDELCELAVDECCEPLECFQMGHGFKRCGSGSSGSS 19 194737 MHHHHHHSGSSSGSGCVKKDELCELAVDECCEPLECTKGDLGFRKCGSGSSGSS 20 194716 MHHHHHHSGSSSGSGCVKKDELCELAIDVCCEPLECLGHGLGYAYCGSGSSGSS n.d. 21 194720 MHHHHHHSGSSSGSGCVKKDELCELAIDVCCEPLECLGHGLGYAYCGSGSSGSS -- -- 11 194328 MHHHHHHSGSSSGSGCVKKDELCNWVTPMRCEPLECLGHGLGYAYCGSGSSGSS n.d. *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 1 197569 MGRGSHHHHHHARSDWELSPPHVAITTRHLINCTDGPLLRDANAPKASAI -- n.d. 2 197536 MGRGSHHHHHHARSLNGESTSNILTTSRKVTEWTGYTASVDANAPKASAI -- n.d. 3 197611 MGRGSHHHHHHARSQVTWHHLADTVTTKNRKCTDSYIGWNXANAPKASAI -- n.d. 4 197530 MGRGSHHHHHHARSIIVIHNAIQTHTPHQVSIWCPPKHNRDANAPKASAI -- n.d. 5 197557 MGRGSHHHHHHARSSHCRHRNCHTITRGNMRIETPNNIRKDANAPKASAI 6 197596 MGRGSHHHHHHARSTMNTNRMDIQRLMTNHVKRDSSPGSIDANAPKASAI 7 197552 MGRGSHHHHHHARSLSALRRTERTWNTIHQGHHLEWYPPADANAPKASAI -- n.d. 8 197541 MGRGSHHHHHHARSWTSMQGETLWRTDRLATTKTSMSHPPDANAPKASAI -- n.d. 9 197588 MGRGSHHHHHHARSNVIPLNEVWYDTGWDRPHRSRLSIDDDANAPKASAI 10 197635 MGPGSHHHHHHARSCLATRNGFV.MNTDRGTYVKRPTVLQDANAPKASAI -- 11 197797 MGRGSHHHHHHARSWGLSGTQTWKITKLATRLHHPEFETNDANAPKASAI -- n.d. 12 197888 MGRGSHHHHHHARSWRWHNWGLSDTVASHPDASNSLNMMYDANAPKASAN -- n.d. 13 197897 MGRGSHHHHHHLDLWGPPSGSPRTRSTTGTSTTSSPSTPGTLTLRRHPH -- n.d 14 197825 MGRGSHHHHHHARSWQPEVKMSSLVDTSQTVGAAVETRTTDANAPKASA -- 15 198000 MGRGSHHHHHHARSWRDTRKLHMRHYFPLAIDSYWDHTLRDANAPKASAI -- 16 197983 MGRGSHHHHHHHRSWTSMQGETLWRTDRLATTKTSMSHPPDANAPKASAI -- n.d. 17 197998 MGRGSHHHHHHHARSPLWYHYNCWDTICLADWLKDRPHGVYDANAPKASA -- n.d. 18 197947 MGRGSHHHHHHARSVGTTIRIAQDTEHYRNVYHKLSQYSRDANAPKASAI -- 19 197954 MGRGSHHHHHHARSVGTTIRIAQDTEHYRNVYHKLSQYSRDANAPKASAI -- n.d. 20 197971 MGRGSHHHHHHARSNVIPLNEVWYDTGWDRPHRSRLSIDDDANAPKASAI -- n.d. *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
[0095]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 gurmarin-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 Peptide Synthesis of Pertussis Toxin Binding Peptides* Selection Clone Seq # Sequence Purity (%) Yield (mg) pp26 5 c 197557 RSSHCRHRNCHTITRGNMRIETPNNIRKDAK 90-95 7.7 pp26 5 n 197557 RSSHCRHRNCHTITRGNMRIETPNNIRKDA 90-95 7.6 pp26 6 n 197596 RSTMNTNRMDIQRLMTNHVKRDSSPGSIDA 90-95 6.3 pp26 9 n 197588 RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA 90-95 5.8 pp26 15 n 198000 RSWRDTRKLHMRHYFPLAIDSYWDHTLRDA 90-95 4.8 gurmarin 9 c 194259 SGCVKKDELCARWDLVCCEPLECIYTSELYATCGK 70 1.0 gurmarin 9 n 194259 SGCVKKDELCARWDLVCCEPLECIYTSELYATCG 80-90 4.0 gurmarin 10 n 194264 SGCVKKDELCELAVDECCEPLECFQMGHGFKRCG 90-95 4.9 gurmarin 15 n 194511 SGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS 90-95 6.3 gurmarin 19 n 194737 SGCVKKDELCELAIDECCEPLECTKGDLGFRKCG 90-95 6.7 *Abbreviation c in the clone name indicates C-terminal biotinylated peptides, abbreviation n indicates N-terminal biotinylated peptides.
[0096]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
[0097]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
1. Construction of GST Fusions For pp26-Variants
[0098]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') and #468 (5'-phosphate-GGGTTAAATAGCGGATGCCTTCGGAGCGTTAGCGTC-3') 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
[0099]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') and #465 (5'-phosphate-GGGTTAAATAGCGGATGCTACTAGGC-3') 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 NdeI/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
[0100]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 PreScission® Protease
[0101]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 160U 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
[0102]Two process fluids were considered as potential starting material for affinity chromatography process:
[0103]Sample A Concentrated culture filtrate containing 10-50 μg/ml (˜0.09-0.45 μM) crude PT, fermentation supernatant
[0104]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 koff 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 Bmax 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 (Bmax) and dissociation constant (KD) of the most suitable binder may also be performed.
TABLE-US-00009 TABLE 7 Overview about fraction of binding active peptides under the applied experimental conditions Peptide pp26 pp26 pp26 gurm gurm gurm gurm name 5n 9n 15n 9n 10n 15n 19n Fraction >5% 1 >50% >12.5% >12.5% |>5% 1 >50% >5% 1 of binding active peptide 1 Calculation 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 glycin 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 sensograms 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-00010 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 hexamer 93 98 100 100 100 100 100 100 98 95 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-00011 TABLE 9 Pertussis toxin hexamer stability under different salt conditions PT hexamer stability pH 5 pH 8.5 NaCl 0-2.5 M 0-2.5 M stable stable KCl 0-2.0 M 0-2.0 M stable stable MgCl2 Nd 0-2.0 M 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 glycin 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 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 glycin, 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-00012 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 3-9 3-9 3-9 3-9 stable, stable, stable, stable, pH range instable at pH 2.5 or instable at pH 2.5 or instable at pH 2.5 or instable at pH 2.5 or stability of 10.5 10.5 10.5 10.5 the complex pH 6 pH 8.5 pH 6 pH 8.5 pH 8.5 pH 6 pH 8.5 NaCl 2 M 2 M 2 M 2 M strong sensitive to salt sensitive to salt stability of stable stable stable stable the complex KCl 2 M 2 M 2 M 2 M strong sensitive to salt sensitive to salt stability of stable stable stable stable the complex MgCl2 stable Sensitive stable up to Elution ≧ 1.5, Elution ≧ 0.5 M Elution ≧ 1 M, Elution ≧ 1 M, stability of from 125 mM, 2 M but not complete complete the complex complete complete elution ≧ elution ≧ elution ≧ 2 M 2 M 2 M
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 glycin, pH 2.5 more gentle for PT hexamer stability than 100 mM carbonat buffer, pH 10.5, see xxx). 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 glycin, 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
a. Purification of PT From Sample B Under Optimized Wash And Elution Conditions (4 μl Column)
[0139]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.
[0140]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 peptide 15, 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).
[0141]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:
[0142](a) with 50 mM glycine, pH 2.5 (each 20 μl) in case of column 1, or
[0143](b) with 100 mM carbonate buffer, pH 10.5 (each 20 μl) in case of column 2, or
[0144](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-00013 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 Glycin pH 2.5 2 pmol PT in 3/120 80 pmol >48% 20% of pooled elutions Carbonat 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 Glycin pH 2.5 2 pmol PT in 3/120 80 pmol >48% 20% of pooled elutions Carbonat 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
[0145]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 streptavidin 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.
[0146]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
[0147]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-00014 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%
[0148]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
[0149]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
[0150]Optimized conditions for PT binding and elution were applied for large scale FPLC purification (0.5 ml column), as shown below:
[0151]A) Immobilization of biotinylated peptide to streptavidin-sepharose: 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.
[0152]B) Binding of PT (out of sample B): 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/μl, corresponding to 12.5 nmol in 25 ml, corresponding to a ratio of immobilized peptide to amount of PT of 4:1).
[0153]C) FPLC-column: 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.
[0154]D) analysis of elution fractions and calculation of yield: 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).
[0155]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-00015 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-00016 TABLE 14 Summary of PT Purification Results Relative Yield versus Yield PT in 12x input amount of PT 0.5 ml (1125 μg in 25 ml) fractions (6 ml) (pmol/pmol or μg/μg) Purity 1. purification run 772-813 μg 69%-72% Comparable to PT purified on asialofetuin sepharose, 100% 2. purification run .sup. 803 μg 71% Comparable to PT purified on asialofetuin sepharose, 100%
6. Evaluation Of Equilibrium And Rate Constants of the pp26 Peptide 9/Pertussis Toxin Complex Formation
[0156]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 immobilized 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:
TABLE-US-00017 Dissociation equilibrium constant KD→ 7.5 × 10-9 M Association equilibrium constant KA→ 1.3 × 10-8 M-1 Association rate constant kon → 1.3 × 105 M-1 × s-1 Dissociation rate constant koff → 10-3 s-1
[0157]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
37417PRTGymnema sylvestre 1Asn Gly Ser Phe Ser Gly Phe1
527PRTGymnema sylvestre 2Asn Gly Ser Phe Ser Gly Cys1
537PRTGymnema sylvestre 3Asp Gly Ser Phe Ser Gly Phe1
547PRTGymnema sylvestreMISC_FEATURE(1)..(7)X is any amino acid 4Xaa Gly
Ser Phe Ser Gly Xaa1 5530PRTGymnema sylvestre 5Arg Ser Ser
His Cys Arg His Arg Asn Cys His Thr Ile Thr Arg Gly1 5
10 15Asn Met Arg Ile Glu Thr Pro Asn Asn
Ile Arg Lys Asp Ala 20 25
30629PRTGymnema sylvestre 6Ser Thr Met Asn Thr Asn Arg Met Asp Ile Gln
Arg Leu Met Thr Asn1 5 10
15His Val Lys Arg Asp Ser Ser Pro Gly Ser Ile Asp Ala 20
25730PRTGymnema sylvestre 7Arg Ser Asn Val Ile Pro Leu Asn
Glu Val Trp Tyr Asp Thr Gly Trp1 5 10
15Asp Arg Pro His Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala
20 25 30830PRTGymnema sylvestre
8Arg Ser Trp Arg Asp Thr Arg Lys Leu His Met Arg His Tyr Phe Pro1
5 10 15Leu Ala Ile Asp Ser Tyr
Trp Asp His Thr Leu Arg Asp Ala 20 25
30934PRTGymnema sylvestre 9Ser Gly Cys Val Lys Lys Asp Glu Leu
Cys Ala Arg Trp Asp Leu Val1 5 10
15Cys Cys Glu Pro Leu Glu Cys Ile Tyr Thr Ser Glu Leu Tyr Ala
Thr 20 25 30Cys
Gly1034PRTGymnema sylvestre 10Ser Gly Cys Val Lys Lys Asp Glu Leu Cys Glu
Leu Ala Val Asp Glu1 5 10
15Cys Cys Glu Pro Leu Glu Cys Phe Gln Met Gly His Gly Phe Lys Arg
20 25 30Cys Gly1135PRTGymnema
sylvestre 11Ser Gly Cys Val Lys Lys Asp Glu Leu Cys Ser Gln Ser Val Pro
Met1 5 10 15Cys Cys Glu
Pro Leu Glu Cys Lys Trp Phe Asn Glu Asn Tyr Gly Ile 20
25 30Cys Gly Ser 351234PRTGymnema
sylvestre 12Ser Gly Cys Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp
Glu1 5 10 15Cys Cys Glu
Pro Leu Glu Cys Thr Lys Gly Asp Leu Gly Phe Arg Lys 20
25 30Cys Gly1335PRTGymnema sylvestre 13Gln Gln
Cys Val Lys Lys Asp Glu Leu Cys Ile Pro Tyr Tyr Leu Asp1 5
10 15Cys Cys Glu Pro Leu Glu Cys Lys
Lys Val Asn Trp Trp Asp His Lys 20 25
30Cys Ile Gly 351431PRTGymnema
sylvestreMISC_FEATURE(9)..(30)X is any amino acid 14Cys Val Lys Lys Asp
Glu Leu Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys1 5
10 15Glu Pro Leu Glu Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys 20 25
3015141DNAGymnema sylvestremisc_feature(49)..(113)n is a, g, t or c
15agtggctcaa gctcaggatc aggctgcgtc aagaaagacg agctctgcnn snnsnnsnns
60nnsnnstgct gtgagcccct cgagtgcnns nnsnnsnnsn nsnnsnnsnn snnstgcggc
120agcggcagtt ctgggtctag c
1411684DNAGymnema sylvestre 16taatacgact cactataggg acaattacta tttacaatta
caatgcacca tcaccatcac 60catagtggct caagctcagg atca
841744DNAGymnema sylvestre 17ttttaaatag
cggatgctac taggctagac ccagaactgc cgct
441810RNAGymnema sylvestre 18uagcggaugc
101953PRTGymnema
sylvestreMISC_FEATURE(18)..(43)Xaa is any amino acid 19Thr Met Val Met
Gly Arg Gly Ser His His His His His His Ala Arg1 5
10 15Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 25
30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Ala Asn Ala Pro
35 40 45Lys Ala Ser Ala Ile
50206PRTArtificial SequenceSynthetic histidine tag 20His His His His His
His1 5216PRTArtificial SequenceSynthetic amino acid linker
21Asp Ala Asn Ala Pro Lys1 522127DNAGymnema
sylvestreMISC_FEATURE(28)..(105)n is A, T, G or C 22agcggatgcc ttcggagcgt
tagcgtcnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnagatc tagcatgatg 120atgatga
1272381DNAGymnema sylvestre
23taatacgact catagggaca attactattt acaattacaa tgggacgtgg ctcacatcat
60catcatcatc atgctagatc t
812432DNAGymnema sylvestre 24aattaaatag cggatgcctt cggagcgtta gc
322518DNABacteriophage M13 25tgtaaaacga cggccagt
182654PRTGymnema
sylvestre 26Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Ala Gly Ser Val Gly His Cys Cys Glu 20
25 30Pro Leu Glu Cys Leu Arg Arg Phe Leu Asn
Leu Arg Trp Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser 502754PRTGymnema sylvestre 27Met His His His
His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Lys Ala Phe
Arg Tyr Ser Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Arg Lys Trp Leu Lys Ala Arg Phe Cys Gly Ser
35 40 45Gly Ser Ser Gly Ser Ser
502854PRTGymnema sylvestre 28Met His His His His His His Ser Gly Ser Ser
Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Lys Ala Phe Arg Tyr Ser Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Arg Lys
Trp Leu Lys Ala Arg Phe Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser 502954PRTGymnema sylvestre 29Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Leu Arg Ser Ser Ile Asp Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Tyr Lys Trp Met Gln Arg Arg Leu Cys
Gly Ser 35 40 45Gly Ser Ser Gly
Ser Ser 503054PRTGymnema sylvestre 30Met His His His His His His Ser
Gly Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Trp Pro Arg Arg His Lys Cys
Cys Glu 20 25 30Pro Leu Glu
Cys Leu Leu Glu Met Leu Glu Arg Lys Arg Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser 503153PRTGymnema
sylvestre 31Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Met Ser Met Ala Cys Val Cys Cys Glu 20
25 30Pro Leu Glu Cys Lys Tyr His Gly Tyr Phe
Trp Leu Cys Gly Ser Gly 35 40
45Ser Ser Gly Ser Ser 503254PRTGymnema sylvestre 32Met His His His His
His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Ala Val Trp Phe
Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Thr Tyr Gln Ser Gly Tyr Tyr Trp Leu Cys Gly Ser
35 40 45Gly Ser Ser Gly Ser Ser
503354PRTGymnema sylvestre 33Met His His His His His His Ser Gly Ser Ser
Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Pro Trp Tyr Trp Arg Cys Cys Glu
20 25 30Pro Leu Glu Cys Val Tyr Thr
Ser Gly Tyr Tyr Tyr Ser Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser 503454PRTGymnema sylvestre 34Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Ala Arg Trp Asp Leu Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Ile Tyr Thr Ser Glu Leu Tyr Ala Thr Cys
Gly Ser 35 40 45Gly Ser Ser Gly
Ser Ser 503554PRTGymnema sylvestre 35Met His His His His His His Ser
Gly Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Val Phe Tyr Phe Pro Asn Cys
Cys Glu 20 25 30Pro Leu Glu
Cys Arg Trp Val Asn Asp Asn Tyr Gly Trp Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser 503653PRTGymnema
sylvestre 36Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Met Ser Met Ala Cys Val Cys Cys Glu 20
25 30Pro Leu Glu Cys Lys Tyr His Gly Tyr Phe
Trp Leu Cys Gly Ser Gly 35 40
45Ser Ser Gly Ser Ser 503754PRTGymnema sylvestre 37Met His His His His
His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Thr Thr Ala Ser
Lys Ser Cys Cys Glu 20 25
30Pro Leu Glu Cys Lys Trp Thr Asn Glu His Phe Gly Thr Cys Gly Ser
35 40 45Gly Ser Ser Gly Ser Ser
503854PRTGymnema sylvestre 38Met His His His His His His Ser Gly Ser Ser
Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Ser Gln Ser Val Pro Met Cys Cys Glu
20 25 30Pro Leu Glu Cys Lys Trp Phe
Asn Glu Asn Tyr Gly Ile Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser 503954PRTGymnema sylvestre 39Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Ala Arg Trp Asp Leu Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Ile Tyr Thr Ser Glu Leu Tyr Ala Thr Cys
Gly Ser 35 40 45Gly Ser Ser Gly
Ser Ser 504054PRTGymnema sylvestre 40Met His His His His His His Ser
Gly Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys
Cys Glu 20 25 30Pro Leu Glu
Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser 504153PRTGymnema
sylvestre 41Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Met Trp Ser Arg Glu Val Cys Cys Glu 20
25 30Pro Leu Glu Cys Tyr Tyr Thr Gly Trp Tyr
Trp Ala Cys Gly Ser Gly 35 40
45Ser Ser Gly Ser Ser 504254PRTGymnema sylvestre 42Met His His His His
His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Val
Asp Glu Cys Cys Glu 20 25
30Pro Leu Glu Cys Phe Gln Met Gly His Gly Phe Lys Arg Cys Gly Ser
35 40 45Gly Ser Ser Gly Ser Ser
504354PRTGymnema sylvestre 43Met His His His His His His Ser Gly Ser Ser
Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Val Asp Glu Cys Cys Glu
20 25 30Pro Leu Glu Cys Thr Lys Gly
Asp Leu Gly Phe Arg Lys Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser 504454PRTGymnema sylvestre 44Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys
Gly Ser 35 40 45Gly Ser Ser Gly
Ser Ser 504554PRTGymnema sylvestre 45Met His His His His His His Ser
Gly Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys
Cys Glu 20 25 30Pro Leu Glu
Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser 504654PRTGymnema
sylvestre 46Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Asn Trp Val Thr Pro Met Arg Cys Glu 20
25 30Pro Leu Glu Cys Leu Gly His Gly Leu Gly
Tyr Ala Tyr Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser 504750PRTGymnema sylvestre 47Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Asp Trp1 5
10 15Glu Leu Ser Pro Pro His Val Ala Ile Thr
Thr Arg His Leu Ile Asn 20 25
30Cys Thr Asp Gly Pro Leu Leu Arg Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 504850PRTGymnema
sylvestre 48Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu
Asn1 5 10 15Gly Glu Ser
Thr Ser Asn Ile Leu Thr Thr Ser Arg Lys Val Thr Glu 20
25 30Trp Thr Gly Tyr Thr Ala Ser Val Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 504950PRTGymnema sylvestreMISC_FEATURE(41)..(41)Xaa is any
amino acid 49Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Gln
Val1 5 10 15Thr Trp His
His Leu Ala Asp Thr Val Thr Thr Lys Asn Arg Lys Cys 20
25 30Thr Asp Ser Tyr Ile Gly Trp Asn Xaa Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 505050PRTGymnema sylvestre 50Met Gly Arg Gly Ser His His His
His His His Ala Arg Ser Ile Ile1 5 10
15Val Ile His Asn Ala Ile Gln Thr His Thr Pro His Gln Val
Ser Ile 20 25 30Trp Cys Pro
Pro Lys His Asn Arg Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 505150PRTGymnema sylvestre 51Met
Gly Arg Gly Ser His His His His His His Ala Arg Ser Ser His1
5 10 15Cys Arg His Arg Asn Cys His
Thr Ile Thr Arg Gly Asn Met Arg Ile 20 25
30Glu Thr Pro Asn Asn Ile Arg Lys Asp Ala Asn Ala Pro Lys
Ala Ser 35 40 45Ala Ile
505250PRTGymnema sylvestre 52Met Gly Arg Gly Ser His His His His His His
Ala Arg Ser Thr Met1 5 10
15Asn Thr Asn Arg Met Asp Ile Gln Arg Leu Met Thr Asn His Val Lys
20 25 30Arg Asp Ser Ser Pro Gly Ser
Ile Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 505350PRTGymnema sylvestre 53Met Gly Arg Gly Ser
His His His His His His Ala Arg Ser Leu Ser1 5
10 15Ala Leu Arg Arg Thr Glu Arg Thr Trp Asn Thr
Ile His Gln Gly His 20 25
30His Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 505450PRTGymnema
sylvestre 54Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp
Thr1 5 10 15Ser Met Gln
Gly Glu Thr Leu Trp Arg Thr Asp Arg Leu Ala Thr Thr 20
25 30Lys Thr Ser Met Ser His Pro Pro Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 505550PRTGymnema sylvestre 55Met Gly Arg Gly Ser His His His
His His His Ala Arg Ser Asn Val1 5 10
15Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg
Pro His 20 25 30Arg Ser Arg
Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 505649PRTGymnema sylvestre 56Met
Gly Arg Gly Ser His His His His His His Ala Arg Ser Cys Leu1
5 10 15Ala Thr Arg Asn Gly Phe Val
Met Asn Thr Asp Arg Gly Thr Tyr Val 20 25
30Lys Arg Pro Thr Val Leu Gln Asp Ala Asn Ala Pro Lys Ala
Ser Ala 35 40
45Ile5750PRTGymnema sylvestre 57Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Trp Gly1 5 10
15Leu Ser Gly Thr Gln Thr Trp Lys Ile Thr Lys Leu Ala Thr Arg Leu
20 25 30His His Pro Glu Phe Glu
Thr Asn Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 505850PRTGymnema sylvestre 58Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Trp Arg1 5
10 15Trp His Asn Trp Gly Leu Ser Asp Thr Val
Ala Ser His Pro Asp Ala 20 25
30Ser Asn Ser Leu Asn Met Met Tyr Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Asn 505949PRTGymnema
sylvestre 59Met Gly Arg Gly Ser His His His His His His Leu Asp Leu Trp
Gly1 5 10 15Pro Pro Ser
Gly Ser Pro Arg Thr Arg Ser Thr Thr Gly Thr Ser Thr 20
25 30Thr Ser Ser Pro Ser Thr Pro Gly Thr Leu
Thr Leu Arg Arg His Pro 35 40
45His6049PRTGymnema sylvestre 60Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Trp Gln1 5 10
15Pro Glu Val Lys Met Ser Ser Leu Val Asp Thr Ser Gln Thr Val Gly
20 25 30Ala Ala Val Glu Thr Arg
Thr Thr Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala6150PRTGymnema sylvestre 61Met Gly Arg Gly Ser His His
His His His His Ala Arg Ser Trp Arg1 5 10
15Asp Thr Arg Lys Leu His Met Arg His Tyr Phe Pro Leu
Ala Ile Asp 20 25 30Ser Tyr
Trp Asp His Thr Leu Arg Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 506250PRTGymnema sylvestre
62Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Thr1
5 10 15Ser Met Gln Gly Glu Thr
Leu Trp Arg Thr Asp Arg Leu Ala Thr Thr 20 25
30Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
506350PRTGymnema sylvestre 63Met Gly Arg Gly Ser His His His His His His
His Ala Arg Ser Pro1 5 10
15Leu Trp Tyr His Tyr Asn Cys Trp Asp Thr Ile Cys Leu Ala Asp Trp
20 25 30Leu Lys Asp Arg Pro His Gly
Val Tyr Asp Ala Asn Ala Pro Lys Ala 35 40
45Ser Ala 506450PRTGymnema sylvestre 64Met Gly Arg Gly Ser
His His His His His His Ala Arg Ser Val Gly1 5
10 15Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His
Tyr Arg Asn Val Tyr 20 25
30His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 506550PRTGymnema
sylvestre 65Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val
Gly1 5 10 15Thr Thr Ile
Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20
25 30His Lys Leu Ser Gln Tyr Ser Arg Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 506650PRTGymnema sylvestre 66Met Gly Arg Gly Ser His His His
His His His Ala Arg Ser Asn Val1 5 10
15Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg
Pro His 20 25 30Arg Ser Arg
Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 506728DNAGymnema sylvestre
67catgccatgg gacgtggctc acatcatc
286836DNAGymnema sylvestre 68gggttaaata gcggatgcct tcggagcgtt agcgtc
366938DNAGymnema sylvestre 69ggagatctca
tatgcaccat caccatcacc atagtggc
387026DNAGymnema sylvestre 70gggttaaata gcggatgcta ctaggc
267154PRTGymnema sylvestre 71Met His His His His
His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Ala Gly Ser Val
Gly His Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Arg Arg Phe Leu Asn Leu Arg Trp Cys Gly Ser
35 40 45Gly Ser Ser Gly Ser Ser
507254PRTGymnema sylvestre 72Met His His His His His His Ser Gly Ser Ser
Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Ile Val Met Arg Ala Pro Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Arg Arg
Tyr Met Leu Lys His Met Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser 507354PRTGymnema sylvestre 73Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Lys Ala Phe Arg Tyr Ser Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Arg Lys Trp Leu Lys Ala Arg Phe Cys
Gly Ser 35 40 45Gly Ser Ser Gly
Ser Ser 507454PRTGymnema sylvestre 74Met His His His His His His Ser
Gly Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Leu Arg Ser Ser Ile Asp Cys
Cys Glu 20 25 30Pro Leu Glu
Cys Leu Tyr Lys Trp Met Gln Arg Arg Leu Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser 507554PRTGymnema
sylvestre 75Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Trp Pro Arg Arg His Lys Cys Cys Glu 20
25 30Pro Leu Glu Cys Leu Leu Glu Met Leu Glu
Arg Lys Arg Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser 507653PRTGymnema sylvestre 76Met His His His
His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Met Ser Met
Ala Cys Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Lys Tyr His Gly Tyr Phe Trp Leu Cys Gly Ser Gly
35 40 45Ser Ser Gly Ser Ser
507754PRTGymnema sylvestre 77Met His His His His His His Ser Gly Ser Ser
Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Ala Val Trp Phe Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Thr Tyr Gln
Ser Gly Tyr Tyr Trp Leu Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser 507854PRTGymnema sylvestre 78Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Glu Pro Trp Tyr Trp Arg Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Tyr Thr Ser Gly Tyr Tyr Tyr Ser Cys
Gly Ser 35 40 45Gly Ser Ser Gly
Ser Ser 507954PRTGymnema sylvestre 79Met His His His His His His Ser
Gly Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys
Cys Glu 20 25 30Pro Leu Glu
Cys Ile Tyr Thr Ser Glu Leu Tyr Ala Thr Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser 508062PRTGymnema
sylvestre 80Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20
25 30Pro Leu Glu Cys Leu Gly His Gly Leu Gly
Tyr Ala Tyr Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50
55 608150PRTGymnema sylvestre 81Met His His His
His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala
Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser
35 40 45Gly Ile 508252PRTGymnema
sylvestre 82Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20
25 30Pro Leu Glu Cys Leu Gly His Gly Leu Gly
Tyr Ala Tyr Cys Gly Ser 35 40
45Gly Ser Ser Gly 508349PRTGymnema sylvestre 83Met His His His His His
His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp
Val Cys Cys Glu 20 25 30Pro
Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35
40 45Gly8458PRTGymnema sylvestre 84Leu His
His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu
Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
Ser 35 40 45Gly Ser Ser Gly Ser
Ser Leu Val Asp Pro 50 558550PRTGymnema sylvestre
85Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu
Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr
Cys Gly Ser 35 40 45Gly Asn
508646PRTGymnema sylvestre 86Met His His His His His His Ser Gly Ser Ser
Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Gly His
Gly Leu Gly Tyr Ala Tyr Cys 35 40
458754PRTGymnema sylvestre 87Met His His His His His His Ser Gly Ser Ser
Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Val Asp Glu Cys Cys Glu
20 25 30Pro Leu Glu Cys Phe Gln Met
Gly His Gly Phe Lys Arg Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Asn 508848PRTGymnema sylvestre 88Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Phe Lys Arg Phe Ser Phe Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys
Gly Ser 35 40 458948PRTGymnema
sylvestre 89Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Phe Lys Arg Phe Ser Phe Cys Cys Glu 20
25 30Pro Leu Glu Cys Leu Gly His Gly Leu Gly
Tyr Ala Tyr Cys Gly Asn 35 40
459062PRTGymnema sylvestre 90Met His His His His His His Ser Gly Ser Ser
Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Trp Ile Arg Phe Val Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Asp Cys Gly
Thr Cys Met Phe Tyr Ser Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50
55 609162PRTGymnema sylvestre 91Met His
His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Ala
Val Trp Phe Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Thr Tyr Gln Ser Gly Tyr Tyr Trp Leu Cys Gly
Ser 35 40 45Gly Ser Ser Gly Ser
Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55
609262PRTGymnema sylvestre 92Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Leu Thr Gln Thr Arg Ser Cys Cys Glu
20 25 30Pro Leu Glu Cys Arg Phe
Leu Arg Ser His Ala Arg Thr Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile
50 55 609362PRTGymnema sylvestre 93Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Arg Lys Arg Tyr Arg Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Ile Leu Gln Phe Met Asn Lys Met Phe Cys
Gly Ser 35 40 45Gly Ser Ser Gly
Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55
609462PRTGymnema sylvestre 94Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Trp1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Pro Trp Tyr Trp Arg Cys Cys
Glu 20 25 30Pro Leu Glu Cys
Val Tyr Thr Ser Gly Tyr Tyr Tyr Ser Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser
Ala Ile 50 55 609562PRTGymnema
sylvestre 95Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Ala Gly Ser Val Gly His Cys Cys Glu 20
25 30Pro Leu Glu Cys Leu Arg Arg Phe Leu Asn
Leu Arg Trp Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50
55 609662PRTGymnema sylvestre 96Met His His His
His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Ala Ser Arg
Ile Trp Ala Cys Cys Gly 20 25
30Pro Leu Glu Cys Leu Met Arg Phe Met Ala Lys Arg Phe Cys Gly Ser
35 40 45Gly Ser Ser Gly Ser Ser Leu Val
Ala Ser Ala Ser Ala Ile 50 55
609752PRTGymnema sylvestre 97Met His His His His His His Ser Gly Ser Ser
Ser Gly Ser Gly Cys1 5 10
15Ala Lys Lys Asp Glu Leu Cys Ser Pro Ala Arg Arg Ile Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Arg Arg
Trp Tyr Glu Glu Ser Phe Cys Gly Ser 35 40
45Gly Ser Ser Gly 509862PRTGymnema sylvestre 98Met His His
His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Thr Met
Asn Glu Val Cys Cys Cys Glu 20 25
30Pro Leu Glu Cys Tyr Gly Asp Ile Ser Gly Glu Ala Met Cys Gly Ser
35 40 45Gly Ser Ser Gly Ser Ser Leu
Val Ala Ser Ala Ser Ala Ile 50 55
609962PRTGymnema sylvestre 99Met His His His His His His Ser Gly Ser Ser
Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Ile Val Met Arg Ala Pro Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Arg Arg
Tyr Met Leu Lys His Met Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50
55 6010062PRTGymnema sylvestre 100Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Lys Ala Phe Arg Tyr Ser Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Arg Lys Trp Leu Lys Ala Arg Phe Cys
Gly Ser 35 40 45Gly Ser Ser Gly
Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55
6010155PRTGymnema sylvestre 101Met His His His His His His Ser
Gly Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Val Ser Gly Leu Met Asn Cys
Cys Glu 20 25 30Pro Leu Glu
Cys Leu Trp Arg Trp Met Gln Lys Gln Gln Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser His 50
5510255PRTGymnema sylvestre 102Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Trp Arg Pro Ala Ile Thr Cys Cys
Glu 20 25 30Pro Leu Glu Cys
Leu Arg Ile Tyr Met Arg Leu Trp Arg Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser Leu 50
5510356PRTGymnema sylvestre 103Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Ser Gln Leu Asp Ser Ala Cys Cys Glu
20 25 30Pro Leu Glu Cys Val Trp
Gln Asn Asp Asn Tyr Gly Thr Cys Gly Arg 35 40
45Ala Val Leu Gly Leu Ala His Pro 50
5510451PRTGymnema sylvestre 104Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Leu Met Arg Val Leu Arg Cys Cys Glu
20 25 30Pro Leu Glu Cys Trp Val
Gly Gly Val Cys Arg Gly Gly Cys Gly Ser 35 40
45Gly Ser Tyr 5010562PRTGymnema sylvestre 105Met His His
His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Thr Lys
Ile Phe Lys Arg Cys Cys Glu 20 25
30Pro Leu Glu Cys Ser Trp Val Val Trp Phe Pro Tyr Ser Cys Gly Ser
35 40 45Gly Ser Ser Gly Ser Ser Leu
Val Ala Ser Ala Ser Ala Ile 50 55
6010662PRTGymnema sylvestre 106Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Lys Lys Ile Asn Ala Lys Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Arg
Arg Phe Leu Arg Phe Lys Phe Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile
50 55 6010762PRTGymnema sylvestre
107Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu
Cys Leu Arg Ser Ser Ile Asp Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Tyr Lys Trp Met Gln Arg Arg Leu
Cys Gly Ser 35 40 45Gly Ser Ser
Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55
6010862PRTGymnema sylvestre 108Met His His His His His His
Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Gly Leu Leu Thr Ser Val
Cys Cys Glu 20 25 30Pro Leu
Glu Cys Val Trp Val Leu His His Phe Val Tyr Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser
Ala Ser Ala Ile 50 55
6010958PRTGymnema sylvestre 109Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Thr Thr Ala Ser Lys Ser Cys Cys Glu
20 25 30Pro Leu Glu Cys Lys Trp
Thr Asn Glu His Phe Gly Thr Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Tyr 50
5511052PRTGymnema sylvestre 110Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys Cys
Glu 20 25 30Pro Leu Glu Cys
Ile Tyr Thr Ser Glu Leu Tyr Ala Thr Cys Gly Ser 35
40 45Gly Ser Ser Gly 5011157PRTGymnema sylvestre
111Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu
Cys Met Ser Met Ala Cys Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Lys Tyr His Gly Tyr Phe Trp Leu Cys
Gly Ser Ala 35 40 45Val Leu Gly
Pro Ser Ser Ile Arg Tyr 50 5511256PRTGymnema
sylvestre 112Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Phe Trp Trp Leu Thr Leu Cys Cys Glu 20
25 30Pro Leu Glu Cys Leu Arg His Ile Cys Leu
Val Ser Pro Cys Gly Arg 35 40
45Ala Val Leu Gly Leu Ala His Pro 50
5511362PRTGymnema sylvestre 113Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Arg Lys Arg Arg Asn Gly His Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Trp
Trp Ala Gly Val Pro Leu Met Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile
50 55 6011462PRTGymnema sylvestre
114Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu
Cys Arg Pro Glu Val Leu Ser Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Arg Arg Trp Phe Gln Lys Arg Met
Cys Gly Ser 35 40 45Gly Ser Ser
Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55
6011562PRTGymnema sylvestre 115Met His His His His His His
Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Phe Ser Arg Met Phe Met
Cys Cys Glu 20 25 30Pro Leu
Glu Cys Asn Cys Pro Leu Ile Met Phe Ile Tyr Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser
Ala Ser Ala Ile 50 55
6011662PRTGymnema sylvestre 116Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Trp Pro Arg Arg His Lys Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Leu
Glu Met Leu Glu Arg Lys Arg Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile
50 55 6011762PRTGymnema sylvestre
117Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu
Cys His Ala Trp Tyr Thr Phe Cys Cys Glu 20 25
30Pro Leu Glu Cys Gln Arg Lys Phe Gly Gly Tyr Trp Ala
Cys Gly Ser 35 40 45Gly Ser Ser
Gly Ser Ser Leu Val Ala Ser Ala Ser Ala Ile 50 55
6011862PRTGymnema sylvestre 118Met His His His His His His
Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Trp Glu Asp Met Thr Val
Cys Cys Glu 20 25 30Pro Leu
Glu Cys Pro Ala Leu Glu Ser Val Val Leu Gln Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser
Ala Ser Ala Ile 50 55
6011950PRTGymnema sylvestre 119Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Leu Cys Trp Gln Trp Thr Cys Cys Glu
20 25 30Pro Leu Glu Cys Glu Leu
Gln Trp Gly Ile Ile Arg Met Cys Gly Ser 35 40
45Gly Asn 5012060PRTGymnema sylvestre 120Met His His His
His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala
Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser
35 40 45Gly Ser Ser Gly Ser Ser Leu Val
Ala Ser Ala Ile 50 55
6012158PRTGymnema sylvestre 121Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Gly
His Gly Leu Gly Tyr Ala Tyr Cys Gly Arg 35 40
45Ala Val Leu Gly Leu Ala His Pro Leu Phe 50
5512260PRTGymnema sylvestre 122Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys
Glu 20 25 30Pro Leu Glu Cys
Phe Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile
50 55 6012359PRTGymnema sylvestre
123Met His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys Val1
5 10 15Lys Lys Asp Glu Leu Cys
Glu Leu Ala Ile Asp Val Cys Cys Glu Pro 20 25
30Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys
Gly Ser Gly 35 40 45Ser Ser Gly
Ser Ser Leu Val Ala Ser Ala Ile 50 5512450PRTGymnema
sylvestre 124Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20
25 30Pro Leu Glu Cys Leu Gly His Gly Leu Gly
Tyr Ala Tyr Cys Gly Ser 35 40
45Gly Asn 5012547PRTGymnema sylvestre 125Met His His His His His His
Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val
Cys Cys Glu 20 25 30Pro Leu
Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35
40 4512646PRTGymnema sylvestre 126Met His His His
His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala
Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys 35
40 4512748PRTGymnema sylvestre 127Met His
His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu
Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
Arg 35 40 4512861PRTGymnema
sylvestre 128Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20
25 30Pro Leu Glu Cys Phe Gly His Gly Leu Gly
Tyr Ala Tyr Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ser Asn 50
55 6012960PRTGymnema sylvestre 129Met His His His His
His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile
Asp Val Cys Cys Glu 20 25
30Pro Phe Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser
35 40 45Gly Ser Ser Gly Ser Ser Leu Val
Ala Ser Ala Ile 50 55
6013060PRTGymnema sylvestre 130Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Gly
His Gly Leu Trp Tyr Ala Tyr Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50
55 6013155PRTGymnema sylvestre 131Met His
His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu
Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
Ser 35 40 45Gly Ser Ser Gly Ser
Ser His 50 5513259PRTGymnema sylvestre 132Met His His
His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Arg Ser Pro Thr Leu
Ser Cys Cys Glu Pro Leu Glu 20 25
30Cys Leu Arg Val Tyr Leu Glu His Trp Phe Cys Gly Ser Gly Ser Gly
35 40 45Ser Ser Leu Val Ala Ser Ala
Ser Ala Ile Asn 50 5513360PRTGymnema sylvestre 133Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Gln Leu Cys Ala Leu His Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Arg Met Met Phe Leu Val His Arg Cys
Gly Ser 35 40 45Gly Ser Ser Gly
Ser Ser Leu Val Ala Ser Ala Ile 50 55
6013461PRTGymnema sylvestre 134Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Asn Trp Val Thr Pro Met Arg Cys Glu
20 25 30Pro Leu Glu Cys Val Leu
Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35 40
45Ser Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50
55 6013561PRTGymnema sylvestre 135Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Val Phe Tyr Phe Pro Asn Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Arg Trp Val Asn Asp Asn Tyr Gly Trp
Cys Gly 35 40 45Ser Gly Ser Ser
Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55
6013661PRTGymnema sylvestre 136Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Thr Thr Ala Ser Lys Ser Cys Cys
Glu 20 25 30Pro Leu Glu Cys
Val Lys Trp Thr Asn Glu His Phe Gly Thr Cys Gly 35
40 45Ser Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala
Ile 50 55 6013758PRTGymnema
sylvestre 137Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Met Ser Met Ala Cys Val Cys Cys Glu 20
25 30Pro Leu Glu Cys Val Lys Tyr His Gly Tyr
Phe Trp Leu Cys Cys Gly 35 40
45Ser Gly Ser Ser Gly Ser Ser Leu Val Glu 50
5513861PRTGymnema sylvestre 138Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Ser Val Trp Tyr Arg Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Val Thr
Pro Asp Trp Ser Gly Ile Leu Tyr Cys Gly 35 40
45Ser Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50
55 6013952PRTGymnema sylvestre 139Ser
Ser Ser Gly Ser Gly Cys Val Lys Lys Asp Glu Leu Cys Glu Leu1
5 10 15Ala Ile Asp Val Cys Cys Glu
Pro Leu Glu Cys Val Leu Gly His Gly 20 25
30Leu Gly Tyr Ala Tyr Cys Gly Ser Gly Ser Ser Gly Ser Ser
Leu Val 35 40 45Ala Ser Ala Ile
5014061PRTGymnema sylvestre 140Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Val Leu
Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35 40
45Ser Val Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50
55 6014159PRTGymnema sylvestre 141Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Leu Gly His Gly Leu Gly Tyr Ala Tyr
Cys Gly 35 40 45Ser Gly Ile Leu
Gly Leu Ala His Pro Leu Phe 50 5514261PRTGymnema
sylvestre 142Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20
25 30Pro Leu Glu Cys Val Leu Gly His Gly Leu
Gly Tyr Ala Tyr Cys Gly 35 40
45Ser Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Val Phe 50
55 6014357PRTGymnema sylvestre 143Met His His His His
His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile
Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
35 40 45Ser Gly Ser Leu Gly Leu Ala His
Pro 50 5514455PRTGymnema sylvestre 144Met His His His
His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala
Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
35 40 45Ser Gly Ser Ser Gly Ser Arg
50 5514554PRTGymnema sylvestre 145Met His His His His
His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile
Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
35 40 45Ser Gly Ser Ser Gly Leu
5014656PRTGymnema sylvestre 146Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Val Leu
Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35 40
45Ser Gly Ser Ser Gly Ser Ser His 50
5514755PRTGymnema sylvestre 147Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Val Leu
Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35 40
45Ser Gly Ser Ser Gly Ser Ser 50
5514855PRTGymnema sylvestre 148Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Val Leu
Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35 40
45Ser Gly Ser Ser Gly Ser Arg 50
5514946PRTGymnema sylvestre 149Ser Gly Ser Ser Ser Gly Ser Gly Cys Val
Lys Lys Asp Glu Leu Cys1 5 10
15Glu Leu Ala Ile Asp Val Cys Cys Glu Pro Leu Glu Cys Val Leu Gly
20 25 30His Gly Leu Gly Tyr Ala
Tyr Cys Gly Ser Gly Ser Ser Gly 35 40
4515053PRTGymnema sylvestre 150Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys
Glu 20 25 30Pro Leu Glu Cys
Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35
40 45Ser Gly Ser Ser Asp 5015152PRTGymnema sylvestre
151Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu
Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Leu Gly His Gly Leu Gly Tyr Ala
Tyr Cys Gly 35 40 45Ser Gly Ser
Ser 5015252PRTGymnema sylvestre 152Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys
Glu 20 25 30Pro Leu Glu Cys
Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35
40 45Ser Gly Ser Tyr 5015352PRTGymnema sylvestre
153Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu
Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Leu Gly His Gly Leu Gly Tyr Ala
Tyr Cys Gly 35 40 45Ser Cys Ser
Tyr 5015451PRTGymnema sylvestre 154Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys
Glu 20 25 30Pro Leu Glu Cys
Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35
40 45Ser Gly Arg 5015550PRTGymnema sylvestre 155Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Leu Gly His Gly Leu Gly Tyr Ala Tyr
Cys Gly 35 40 45Thr Ala
5015646PRTGymnema sylvestre 156Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Val Leu
Gly His Gly Leu Gly Tyr Ala Tyr 35 40
4515751PRTGymnema sylvestre 157Met His His His Ser Gly Ser Ser Ser
Gly Ser Gly Cys Val Lys Lys1 5 10
15Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu Pro Leu
Glu 20 25 30Cys Trp Leu Gly
His Gly Leu Gly Tyr Ala His Cys Gly Ser Gly Ser 35
40 45Ser Gly Ser 5015861PRTGymnema sylvestre 158Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Glu Leu Ala Ile Asp Glu Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Thr Lys Gly Asp Leu Gly Phe Arg Lys
Cys Gly 35 40 45Ser Gly Ser Ser
Gly Ser Ser Leu Val Ala Ser Ala Ile 50 55
6015951PRTGymnema sylvestre 159Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys Cys
Glu 20 25 30Pro Leu Glu Cys
Val Thr Lys Gly Asp Leu Gly Phe Arg Lys Cys Gly 35
40 45Ser Gly Ser 5016051PRTGymnema sylvestre 160Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Ala Arg Trp Asp Leu Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Thr Lys Gly Asp Leu Gly Phe Arg Lys
Cys Gly 35 40 45Ser Gly Tyr
5016155PRTGymnema sylvestre 161Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Ala Arg Trp Asp Leu Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Val Ile
Tyr Thr Ser Glu Leu Tyr Ala Thr Cys Gly 35 40
45Ser Gly Ser Ser Gly Ser Arg 50
5516261PRTGymnema sylvestre 162Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Thr Thr Ala Ser Lys Ser Cys Cys Glu
20 25 30Pro Leu Glu Cys Val Lys
Trp Thr Asn Glu His Phe Gly Thr Cys Gly 35 40
45Ser Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50
55 6016359PRTGymnema sylvestre 163Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Thr Thr Ala Ser Lys Ser Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Lys Trp Thr Asn Glu His Phe Gly Thr
Cys Gly 35 40 45Thr Ala Val Leu
Gly Leu Ala His Pro Leu Phe 50 5516461PRTGymnema
sylvestre 164Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Ser Gln Ser Val Pro Met Cys Cys Glu 20
25 30Pro Leu Glu Cys Val Lys Trp Phe Asn Glu
Asn Tyr Gly Ile Cys Gly 35 40
45Ser Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50
55 6016561PRTGymnema sylvestre 165Met His His His His
His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile
Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
35 40 45Ser Gly Ser Ser Gly Ser Ser Leu
Val Ala Ser Ala Ile 50 55
6016661PRTGymnema sylvestre 166Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Asp
20 25 30Pro Leu Glu Cys Val Leu
Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35 40
45Ser Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50
55 6016761PRTGymnema sylvestre 167Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Ala Lys Lys Asp Glu Leu Cys
Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Leu Gly His Gly Leu Gly Tyr Ala Tyr
Cys Gly 35 40 45Ser Gly Ser Ser
Gly Ser Ser Leu Val Ala Ile Arg Tyr 50 55
6016860PRTGymnema sylvestre 168Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys
Glu 20 25 30Pro Leu Glu Cys
Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35
40 45Ser Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala
50 55 6016959PRTGymnema sylvestre
169Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu
Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Leu Gly His Gly Leu Gly Tyr Ala
Tyr Cys Gly 35 40 45Ser Ala Val
Leu Gly Leu Ala His Pro Leu Phe 50 5517059PRTGymnema
sylvestre 170Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20
25 30Pro Leu Glu Cys Val Leu Gly His Gly Leu
Gly Tyr Ala Tyr Cys Gly 35 40
45Ser Gly Val Leu Gly Leu Ala His Pro Leu Phe 50
5517158PRTGymnema sylvestre 171Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Val Leu
Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35 40
45Ser Gly Ser Ser Gly Ser Ser Leu Val Ala 50
5517256PRTGymnema sylvestre 172Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys
Asp 20 25 30Pro Leu Glu Cys
Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35
40 45Ser Gly Ser Ser Gly Ser Ser His 50
5517361PRTGymnema sylvestre 173Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys
Asp 20 25 30Pro Leu Glu Cys
Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35
40 45Ser Gly Ser Ser Gly Ser Ser Pro Ser Ser Ile Arg
Tyr 50 55 6017459PRTGymnema
sylvestre 174Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Asp 20
25 30Pro Leu Glu Cys Val Leu Gly His Gly Leu
Gly Tyr Ala Tyr Cys Gly 35 40
45Thr Ala Val Leu Gly Leu Ala His Pro Leu Phe 50
5517559PRTGymnema sylvestre 175Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Asp
20 25 30Pro Leu Glu Cys Val Leu
Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35 40
45Ser Gly Ser Leu Gly Leu Ala His Pro Leu Phe 50
5517659PRTGymnema sylvestre 176Met His His His His His His Ser
Gly Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Arg Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys
Cys Asp 20 25 30Pro Leu Glu
Cys Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35
40 45Ser His Ser Ser Gly Leu Ala His Pro Leu Phe
50 5517754PRTGymnema sylvestre 177Met His His His His
His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile
Asp Val Cys Cys Asp 20 25
30Pro Leu Glu Cys Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
35 40 45Ser Gly Ser Ser Gly Ser
5017859PRTGymnema sylvestre 178Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Asp
20 25 30Pro Leu Glu Cys Val Leu
Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35 40
45Arg Ala Val Leu Gly Leu Ala His Pro Leu Phe 50
5517959PRTGymnema sylvestre 179Met His His His His His His Ser
Gly Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys
Cys Asp 20 25 30Pro Leu Glu
Cys Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35
40 45Ser Gly Ser Ser Gly Ser Ser Leu Val Ala Ser
50 5518059PRTGymnema sylvestre 180Met His His His His
His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile
Asp Val Cys Cys Asp 20 25
30Pro Leu Glu Cys Val Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
35 40 45Thr Ala Val Leu Gly Leu Ala His
Pro Leu Phe 50 5518161PRTGymnema sylvestre 181Met His
His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Thr
Thr Ala Ser Lys Ser Cys Cys Glu 20 25
30Pro Leu Glu Cys Val Lys Trp Thr Asn Glu His Phe Gly Thr Cys
Gly 35 40 45Ser Gly Ser Ser Gly
Ser Ser Leu Val Ala Ser Ala Ile 50 55
6018261PRTGymnema sylvestre 182Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Gly
His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile Tyr 50
55 6018351PRTGymnema sylvestre 183Met
His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu Cys
Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys
Gly Ser 35 40 45Gly Ser Tyr
5018457PRTGymnema sylvestreMISC_FEATURE(57)..(57)Xaa is any amino acid
184Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu
Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr
Cys Gly Ser 35 40 45Gly Ser Ser
Gly Ser Ser Leu Glu Xaa 50 5518561PRTGymnema
sylvestre 185Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20
25 30Pro Leu Glu Cys Leu Gly His Gly Leu Gly
Tyr Ala Tyr Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Arg Tyr 50
55 6018647PRTGymnema sylvestre 186Met His His His His
His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile
Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly 35
40 4518756PRTGymnema sylvestre 187Met His
His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu
Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
Ser 35 40 45Gly Ser Ser Gly Ser
Ser Leu Asp 50 5518854PRTGymnema sylvestre 188Met His
His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu
Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
Ser 35 40 45Gly Val Leu Gly Leu
Ala 5018959PRTGymnema sylvestre 189Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys
Glu 20 25 30Pro Leu Glu Cys
Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala 50
5519061PRTGymnema sylvestre 190Met His His His His His
His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp
Glu Cys Cys Glu 20 25 30Pro
Leu Glu Cys Thr Lys Gly Asp Leu Gly Phe Arg Lys Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser Leu Val Ala
Ser Ala Ile Tyr 50 55
6019159PRTGymnema sylvestre 191Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Gly
His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40
45Glu Phe Trp Val Pro Ser Ser Ile Arg Tyr Leu 50
5519258PRTGymnema sylvestre 192Met His His His His His His Ser
Gly Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys
Cys Glu 20 25 30Pro Leu Glu
Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser 50
5519353PRTGymnema sylvestre 193Met His His His His His
His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp
Val Cys Cys Glu 20 25 30Pro
Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35
40 45Gly Ser Ser Gly Ser
5019455PRTGymnema sylvestre 194Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Gly
His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser His 50
5519558PRTGymnema sylvestre 195Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Gly
His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40
45Ala Val Leu Gly Leu Ala His Pro Leu Phe 50
5519654PRTGymnema sylvestre 196Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys
Glu 20 25 30Pro Leu Glu Cys
Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35
40 45Gly Ile Leu Gly Leu Ala 5019760PRTGymnema
sylvestre 197Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20
25 30Pro Leu Glu Cys Leu Gly His Gly Leu Gly
Tyr Ala Tyr Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Asn 50
55 6019857PRTGymnema sylvestre 198Met His His His His
His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile
Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser
35 40 45Gly Ser Ser Gly Ser Ser Leu Val
Ala 50 5519952PRTGymnema sylvestre 199Met His His His
His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala
Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser
35 40 45Gly Ser Ser Asp
5020060PRTGymnema sylvestre 200Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Gly
His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Phe 50
55 6020156PRTGymnema sylvestre 201Met His
His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu
Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
Ser 35 40 45Gly Ser Ser Gly Ser
Ser Leu Val 50 5520258PRTGymnema sylvestre 202Met His
His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu
Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
Ser 35 40 45Gly Ser Ser Gly Leu
Ala His Pro Leu Phe 50 5520360PRTGymnema sylvestre
203Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu
Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr
Cys Gly Ser 35 40 45Gly Ser Ser
Gly Ser Ser Leu Val Ala Ser Ala Asn 50 55
6020450PRTGymnema sylvestre 204Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys
Glu 20 25 30Pro Leu Glu Cys
Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35
40 45Gly Arg 5020558PRTGymnema sylvestre 205Met His
His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu
Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
Ser 35 40 45Gly Ser Ser Gly Ser
Ser Leu Val Ala Tyr 50 5520659PRTGymnema sylvestre
206Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1
5 10 15Val Lys Lys Asp Glu Leu
Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr
Cys Gly Ser 35 40 45Gly Ser Ser
Gly Ser Ser Leu Val Ala Ser Ala 50 5520760PRTGymnema
sylvestre 207Met His His His His His His Ser Gly Ser Ser Ser Gly Ser Gly
Cys1 5 10 15Val Lys Lys
Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu 20
25 30Pro Leu Glu Cys Leu Gly His Gly Leu Gly
Tyr Ala Tyr Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Asn 50
55 6020859PRTGymnema sylvestre 208Met His His His His
His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Gly Glu Leu Cys Glu Leu Ala Ile
Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser
35 40 45Gly Ser Ser Gly Ser Ser Leu Val
Ala Ser Ala 50 5520955PRTGymnema sylvestre 209Met His
His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu
Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
Ser 35 40 45Gly Ser Ser Gly Ser
Ser Leu 50 5521058PRTGymnema sylvestre 210Met His His
His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu Leu
Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser
35 40 45Gly Ser Leu Gly Leu Ala His
Pro Leu Tyr 50 5521160PRTGymnema sylvestre 211Met His
His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu
Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
Ser 35 40 45Gly Ser Ser Gly Ser
Ser Leu Val Ala Ser Ala Ile 50 55
6021260PRTGymnema sylvestre 212Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Glu Leu Ala Ile Asp Val Cys Cys Glu
20 25 30Pro Leu Glu Cys Leu Gly
His Gly Leu Gly Tyr Ala Tyr Cys Gly Ser 35 40
45Gly Ser Ser Gly Ser Ser Leu Val Ala Ser Arg Tyr 50
55 6021356PRTGymnema sylvestre 213Met His
His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Glu
Leu Ala Ile Asp Val Cys Cys Glu 20 25
30Pro Leu Glu Cys Leu Gly His Gly Leu Gly Tyr Ala Tyr Cys Gly
Ser 35 40 45Gly Ser Ser Gly Ser
Ser Leu Val 50 5521460PRTGymnema sylvestre 214Met His
His His His His His Ser Gly Ser Ser Ser Gly Ser Gly Cys1 5
10 15Val Lys Lys Asp Glu Leu Cys Met
Trp Ser Arg Glu Val Cys Cys Glu 20 25
30Leu Leu Glu Cys Tyr Tyr Thr Gly Trp Tyr Trp Ala Cys Gly Ser
Gly 35 40 45Ser Ser Gly Ser Ser
Leu Val Ala Ser Ala Ile Tyr 50 55
6021558PRTGymnema sylvestre 215Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Ala Ala1 5 10
15Ser Arg Lys Thr Ser Ser Ala Ser Trp Arg Ser Thr Cys Ala Val Ser
20 25 30Pro Ser Ser Ala Trp Gly
Thr Ala Trp Gly Thr Arg Thr Ala Ala Ala 35 40
45Ala Val Leu Gly Leu Ala His Pro Leu Phe 50
5521659PRTGymnema sylvestre 216Met His His His His His His Ser Gly
Ser Ser Ser Gly Ser Ala Ala1 5 10
15Ser Arg Lys Thr Ser Ser Ala Ser Trp Arg Ser Thr Cys Ala Val
Ser 20 25 30Pro Ser Ser Ala
Trp Gly Thr Ala Trp Gly Thr Arg Thr Ala Ala Ala 35
40 45Ala Val Leu Gly Leu Ala His Pro Pro Ile Tyr 50
5521756PRTGymnema sylvestre 217Met His His His His His
His Ser Gly Ser Ser Ser Gly Ser Ala Ala1 5
10 15Ser Arg Lys Thr Ser Ser Ala Ser Trp Arg Ser Thr
Cys Ala Val Ser 20 25 30Pro
Ser Ser Ala Trp Gly Thr Ala Trp Gly Thr Arg Thr Ala Ala Ala 35
40 45Ala Val Leu Gly Leu Ala His His 50
5521858PRTGymnema sylvestre 218Met His His His His His
His Ser Gly Ser Ser Ser Gly Ser Leu Arg1 5
10 15Gln Glu Arg Arg Ala Leu Arg Ala Gly Asp Arg Arg
Val Leu Ala Pro 20 25 30Arg
Val Leu Gly Ala Arg Pro Gly Val Arg Val Leu Arg Gln Arg Gln 35
40 45Phe Trp Val Pro Ser Ser Ile Arg Tyr
Leu 50 5521954PRTGymnema sylvestre 219Met His His His
His His His Ser Gly Ser Ser Ser Gly Ser Leu Arg1 5
10 15Gln Glu Arg Arg Ala Leu Arg Ala Gly Asp
Arg Arg Val Leu Ala Pro 20 25
30Arg Val Leu Gly Ala Arg Pro Gly Val Arg Val Leu Arg Gln Arg Gln
35 40 45Phe Trp Val Pro Ser Ser
5022059PRTGymnema sylvestre 220Met His His His His His His Ser Gly Ser
Ser Ser Gly Ser Gly Cys1 5 10
15Val Lys Lys Asp Glu Leu Cys Met Trp Ser Arg Glu Val Cys Cys Glu
20 25 30Leu Leu Glu Cys Tyr Tyr
Thr Gly Trp Tyr Trp Ala Cys Gly Ser Gly 35 40
45Ser Ser Gly Ser Ser Leu Val Ala Ser Ala Ile 50
5522150PRTGymnema sylvestre 221Met Gly Arg Gly Ser His His His
His His His Ala Arg Ser Asn Val1 5 10
15Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg
Pro His 20 25 30Arg Ser Arg
Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5022250PRTGymnema sylvestre 222Met
Gly Arg Gly Ser His His His His His His Ala Arg Ser Val Gly1
5 10 15Thr Thr Ile Arg Ile Ala Gln
Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25
30His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys
Ala Ser 35 40 45Ala Ile
5022350PRTGymnema sylvestre 223Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Trp Arg1 5 10
15Asp Thr Arg Lys Leu His Met Arg His Tyr Phe Pro Leu Ala Ile Asp
20 25 30Ser Tyr Trp Asp His Thr
Leu Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5022450PRTGymnema sylvestre 224Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Trp Thr1 5
10 15Ser Met Gln Gly Glu Thr Leu Trp Arg Thr
Asp Arg Leu Ala Thr Thr 20 25
30Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5022550PRTGymnema
sylvestre 225Met Gly Arg Gly Ser His His His His His His His Ala Arg Ser
Pro1 5 10 15Leu Trp Tyr
His Tyr Asn Cys Trp Asp Thr Ile Cys Leu Ala Asp Trp 20
25 30Leu Lys Asp Arg Pro His Gly Val Tyr Asp
Ala Asn Ala Pro Lys Ala 35 40
45Ser Ala 5022650PRTGymnema sylvestre 226Met Gly Arg Gly Ser His His
His His His His Ala Arg Ser Cys Leu1 5 10
15Ala Thr Arg Asn Gly Phe Val Gln Met Asn Thr Asp Arg
Gly Thr Tyr 20 25 30Val Lys
Arg Pro Thr Val Leu Gln Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5022750PRTGymnema sylvestre
227Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Ser1
5 10 15Ala Leu Arg Arg Thr Glu
Arg Thr Trp Asn Thr Ile His Gln Gly His 20 25
30His Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5022850PRTGymnema sylvestre 228Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Thr Met1 5 10
15Asn Thr Asn Arg Met Asp Ile Gln Arg Leu Met Thr Asn His Val Lys
20 25 30Arg Asp Ser Ser Pro Gly
Ser Ile Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5022950PRTGymnema sylvestre 229Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Asp Trp1 5
10 15Glu Leu Ser Pro Pro His Val Ala Ile Thr
Thr Arg His Leu Ile Asn 20 25
30Cys Thr Asp Gly Pro Leu Leu Arg Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5023050PRTGymnema
sylvestre 230Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu
Asn1 5 10 15Gly Glu Ser
Thr Ser Asn Ile Leu Thr Thr Ser Arg Lys Val Thr Glu 20
25 30Trp Thr Gly Tyr Thr Ala Ser Val Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5023150PRTGymnema sylvestreMISC_FEATURE(41)..(41)Xaa is any
amino acid 231Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Gln
Val1 5 10 15Thr Trp His
His Leu Ala Asp Thr Val Thr Thr Lys Asn Arg Lys Cys 20
25 30Thr Asp Ser Tyr Ile Gly Trp Asn Xaa Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5023250PRTGymnema sylvestre 232Met Gly Arg Gly Ser His His
His His His His Ala Arg Ser Ser His1 5 10
15Cys Arg His Arg Asn Cys His Thr Ile Thr Arg Gly Asn
Met Arg Ile 20 25 30Glu Thr
Pro Asn Asn Ile Arg Lys Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5023350PRTGymnema sylvestre
233Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Gly1
5 10 15Leu Ser Gly Thr Gln Thr
Trp Lys Ile Thr Lys Leu Ala Thr Arg Leu 20 25
30His His Pro Glu Phe Glu Thr Asn Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5023450PRTGymnema sylvestre 234Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Trp Arg1 5 10
15Trp His Asn Trp Gly Leu Ser Asp Thr Val Ala Ser His Pro Asp Ala
20 25 30Ser Asn Ser Leu Asn Met
Met Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Asn 5023549PRTGymnema sylvestre 235Met Gly Arg Gly
Ser His His His His His His Leu Asp Leu Trp Gly1 5
10 15Pro Pro Ser Gly Ser Pro Arg Thr Arg Ser
Thr Thr Gly Thr Ser Thr 20 25
30Thr Ser Ser Pro Ser Thr Pro Gly Thr Leu Thr Leu Arg Arg His Pro
35 40 45His23649PRTGymnema sylvestre
236Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp Gln1
5 10 15Pro Glu Val Lys Met Ser
Ser Leu Val Asp Thr Ser Gln Thr Val Gly 20 25
30Ala Ala Val Glu Thr Arg Thr Thr Asp Ala Asn Ala Pro
Lys Ala Ser 35 40
45Ala23750PRTGymnema sylvestre 237Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Trp Thr1 5 10
15Ser Met Gln Gly Glu Thr Leu Trp Arg Thr Asp Arg Leu Ala Thr Thr
20 25 30Lys Thr Ser Met Ser His
Pro Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5023850PRTGymnema sylvestre 238Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Leu Ser1 5
10 15Ala Leu Arg Arg Thr Glu Arg Thr Trp Asn
Thr Ile His Gln Gly His 20 25
30His Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5023950PRTGymnema
sylvestre 239Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu
Ser1 5 10 15Ala Leu Arg
Arg Thr Glu Arg Thr Trp Asn Thr Ile His Gln Gly His 20
25 30His Leu Glu Trp Tyr Pro Thr Ala Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5024050PRTGymnema sylvestre 240Met Gly Arg Gly Ser His His
His His His His Ala Arg Ser Lys Asp1 5 10
15Thr Ala Arg Thr Thr Ala Thr Leu Leu Thr Asn Asp Glu
Asp Arg Lys 20 25 30Thr His
Trp Arg Met Phe Tyr Pro Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5024150PRTGymnema sylvestre
241Met Gly Arg Gly Ser His His His His Tyr His Ala Arg Ser Lys Asp1
5 10 15Thr Ala Arg Thr Thr Ala
Thr Leu Leu Thr Asn Asp Glu Asp Arg Lys 20 25
30Thr His Trp Arg Met Phe Tyr Pro Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5024250PRTGymnema sylvestre 242Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Thr Pro1 5 10
15Arg Leu Arg Lys Val Tyr Asp Leu Thr Val Thr Thr Thr Ser Ser Gln
20 25 30Ile Asp Lys Leu Gln Pro
Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5024350PRTGymnema sylvestre 243Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Ser His1 5
10 15Cys Arg His Arg Asn Cys His Thr Ile Thr
Arg Gly Asn Met Arg Ile 20 25
30Glu Thr Pro Asn Asn Ile Arg Lys Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5024450PRTGymnema
sylvestre 244Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asp
Trp1 5 10 15Glu Leu Ser
Pro Pro His Val Ala Ile Thr Thr Arg His Leu Ile Asn 20
25 30Cys Thr Asp Gly Pro Leu Leu Arg Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5024550PRTGymnema sylvestre 245Met Gly Arg Gly Ser His His
His His His His Ala Arg Ser Ile Ser1 5 10
15Leu Ala Gln Tyr Tyr Trp Thr Ala Gln Arg Asp Met His
Leu Leu Ile 20 25 30Met His
Lys Phe Met Asp Met Pro Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5024650PRTGymnema sylvestre
246Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ile Ile1
5 10 15Val Ile His Asn Ala Ile
Gln Thr His Thr Pro His Gln Val Ser Ile 20 25
30Trp Cys Pro Pro Lys His Asn Arg Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5024750PRTGymnema sylvestre 247Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Lys Phe1 5 10
15Arg Gln Ile Trp Glu Asn Glu Arg Lys Ala His Arg Met Val Met His
20 25 30Gln Phe Tyr Gln Val Ile
Arg Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5024850PRTGymnema sylvestre 248Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Val Ile1 5
10 15Val Cys Val Cys Thr Thr Ala Gly Asn Tyr
Asn His His Asp Gly Phe 20 25
30Phe Lys Arg Tyr Asp Asn Ser Tyr Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5024950PRTGymnema
sylvestre 249Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu
Asn1 5 10 15Gly Glu Ser
Thr Ser Asn Ile Leu Thr Thr Ser Arg Lys Val Thr Glu 20
25 30Trp Thr Gly Tyr Thr Ala Ser Val Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5025050PRTGymnema sylvestre 250Ile Gly Arg Gly Ser His His
His His His His Ala Arg Ser Ser Tyr1 5 10
15Pro Asp His Gly Arg Tyr Arg Asn Gln Ile Glu Arg Gly
Thr Ile Glu 20 25 30Met Thr
Tyr Ile Asp Thr His Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5025149PRTGymnema sylvestre
251Met Gly Arg Gly Ser His His His His His Ala Arg Ser Gly Ala Glu1
5 10 15Pro Gly Met Ser Gly Lys
Pro Lys Val Thr Thr Trp His His Lys Arg 20 25
30Tyr Arg Arg Phe Met Thr His Asp Ala Asn Ala Pro Lys
Ala Ser Ala 35 40
45Ile25246PRTGymnema sylvestre 252Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Asp Ile1 5 10
15Asp Thr Ala Glu Val Asn Arg Trp Glu Ser Asn Leu Lys Ser Tyr Leu
20 25 30Tyr Asn Met Thr Asp Ala
Asn Ala Pro Lys Ala Ser Ala Ile 35 40
4525350PRTGymnema sylvestre 253Met Gly Arg Gly Ser His His His His
His His Ala Arg Ser Val Leu1 5 10
15Thr Gly Gln Ser Leu Tyr Tyr Gln Phe Met Ser Arg Ala Phe Phe
Thr 20 25 30Leu Gln Lys Phe
Thr Gln Asn Leu Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5025450PRTGymnema sylvestre 254Met Gly
Arg Gly Ser His His His His His His Ala Arg Ser Lys Ile1 5
10 15Ala Glu Tyr Trp Leu Thr Glu Arg
Met Met His Leu Arg Ala Met Met 20 25
30Lys Leu Leu Asn Lys His Ala His Asp Ala Asn Ala Pro Lys Ala
Ser 35 40 45Ala Ile
5025550PRTGymnema sylvestre 255Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser His Ser1 5 10
15Ala Leu Met His Asp Lys Asp Ser Ser Thr Ser Thr Tyr Tyr Pro Gln
20 25 30Tyr Ala Asn Ser Pro Ser
Val Gly Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5025650PRTGymnema sylvestre 256Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser His Leu1 5
10 15Asp Pro Cys Ala Asp Leu Asn Val Thr Gln
Gln Arg Thr Thr Arg Glu 20 25
30Thr His Ser Asp Asn Glu Asn His Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5025750PRTGymnema
sylvestre 257Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Pro
Leu1 5 10 15Tyr Gln Gly
Glu Thr Leu Asn Ala Tyr Ala Pro Gln Ser Met Val Lys 20
25 30Ile Ser Lys Asp Tyr Val Leu His Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5025850PRTGymnema sylvestre 258Met Gly Arg Gly Ser His His
His His His His Ala Arg Ser Tyr Met1 5 10
15Ala Arg Trp His Pro Met Thr His Asn His Met Lys Glu
Thr Leu Phe 20 25 30Ala Ala
Glu Pro His Val Cys Thr Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5025950PRTGymnema sylvestre
259Met Gly Arg Gly Ser His His His His His His Ala Arg Pro Arg Phe1
5 10 15His Pro Pro Phe Leu Arg
Asp Arg Ser Val Asn Arg Met Ile Met Asn 20 25
30Glu His Arg Pro Arg Tyr Ser His Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5026050PRTGymnema sylvestre 260Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Ser Pro1 5 10
15Arg Tyr Ala Tyr Cys Gly Ser Arg Trp Asn Gly Ser Arg Met His Asn
20 25 30Asn Lys Phe Thr Pro Ser
Thr Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5026149PRTGymnema sylvestre 261Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Asn Met1 5
10 15Asn Gln Met Thr Asn Ala Leu Asn Leu Arg
Arg Arg Ser Arg Thr Trp 20 25
30Val Ala Thr Phe Arg Ser Glu Asp Ala Asn Ala Pro Lys Ala Ser Ala
35 40 45Ile26250PRTGymnema sylvestre
262Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Met Asn1
5 10 15Gly Leu Asp Met Gly Ser
Pro Ile Trp Tyr Asn Met Gln Leu Lys Leu 20 25
30Ile Tyr Phe Ser Cys Asn Trp Asn Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5026350PRTGymnema sylvestre 263Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Arg Val1 5 10
15Arg Asp Pro Asp Ser Gly Arg Thr His Gln Ile Arg Ser His Leu Lys
20 25 30His Tyr Ser Asn Phe Pro
Val Ala Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5026450PRTGymnema
sylvestreMISC_FEATURE(41)..(41)Xaa is any amino acid 264Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Gln Val1 5
10 15Thr Trp His His Leu Ala Asp Thr Val Thr
Thr Lys Asn Arg Lys Cys 20 25
30Thr Asp Ser Tyr Ile Gly Trp Asn Xaa Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5026548PRTGymnema
sylvestre 265Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ile
Leu1 5 10 15Asp Val Asn
Asp Glu Lys Arg Pro Pro Gly Trp Tyr Arg Thr Asn Ile 20
25 30Ile Asp Ser Pro Ser Gly Asp Ala Asn Ala
Pro Lys Ala Ser Ala Ile 35 40
4526650PRTGymnema sylvestre 266Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Arg Arg1 5 10
15Tyr Arg Asp Gly Ile Phe Arg Arg Met Arg Ser Asx Thr Asn Ala Arg
20 25 30Gly Ala Arg His Ala Asp
Leu Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5026750PRTGymnema sylvestre 267Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Lys Cys1 5
10 15His Val Arg Arg Lys Glu Ser Ala Ser Ser
Lys Asn Arg His Asn His 20 25
30Thr Trp His Asp Ser Asn Leu Tyr Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5026850PRTGymnema
sylvestre 268Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Arg
Thr1 5 10 15Leu Leu Ile
Arg Leu Tyr Pro Pro Asp Arg Phe Gly Ser Ser Arg Gln 20
25 30Met Ala Thr Arg Asp Ser Phe Thr Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5026950PRTGymnema sylvestre 269Met Gly Arg Gly Ser His His
His His His His Ala Arg Ser Ser Gly1 5 10
15Met Tyr Val Val Ser Lys Pro Ala Ser Asp Ser Trp Thr
Thr Cys Ala 20 25 30Pro Tyr
Thr Tyr Gly Thr Met Val Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5027050PRTGymnema sylvestre
270Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Leu1
5 10 15Ser Thr Ile Arg Asx Met
Asn Arg His Leu Thr Asp Arg Arg Leu Thr 20 25
30Ala Phe Arg Asn Gln Val Val Phe Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5027150PRTGymnema sylvestre 271Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Ile Asn1 5 10
15Ala Trp Trp Tyr His Ile Gln Ser His Leu His Gln Trp Arg Arg His
20 25 30Arg Leu Tyr Thr Ala Asn
Gln Trp Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5027250PRTGymnema sylvestre 272Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Thr Met1 5
10 15Asn Thr Asn Arg Met Asp Ile Gln Arg Leu
Met Thr Asn His Val Lys 20 25
30Arg Asp Ser Ser Pro Gly Ser Ile Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5027350PRTGymnema
sylvestre 273Met Gly Arg Gly Ser His His His His His His Ala Arg Pro Asn
Val1 5 10 15Ile Pro Leu
Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20
25 30Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5027450PRTGymnema sylvestre 274Met Gly Arg Gly Ser His His
His His His Arg Ala Arg Ser Asn Val1 5 10
15Ile Pro Leu Ser Glu Val Trp Tyr Asp Thr Gly Trp Asp
Arg Pro His 20 25 30Arg Ser
Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Thr 35
40 45Ala Ile 5027550PRTGymnema sylvestre
275Ile Glu Arg Gly Ser Gln His His His His His Ala Arg Pro Asn Val1
5 10 15Ile Thr Leu Asn Glu Val
Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25
30Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro
Lys Ala Thr 35 40 45Ala Ile
5027649PRTGymnema sylvestre 276Met Gly Arg Gly Ser His His His His His
His Ala Arg Pro Asn Val1 5 10
15Ile Thr Leu Ser Glu Val Trp Asp Thr Gly Trp Asn Arg Pro Leu Arg
20 25 30Gln Arg Cys Arg Ser Glu
Thr Asp Asp Asn Ala Gln Lys Ala Asn Asp 35 40
45Ile27750PRTGymnema sylvestre 277Met Gly Arg Gly Ser His
His His His His His Ala Arg Ser Val Gly1 5
10 15Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr
Arg Asn Val Tyr 20 25 30His
Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5027850PRTGymnema sylvestre
278Met Gly Arg Gly Ser Tyr His His His His His Ala Arg Ser Val Gly1
5 10 15Thr Thr Ile Arg Ile Ala
Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20 25
30His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5027950PRTGymnema sylvestre 279Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Val Gly1 5 10
15Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr
20 25 30His Lys Leu Ser Gln Tyr
Cys Arg Asn Ala Asn Ala Pro Lys Ala Thr 35 40
45Ala Ile 5028050PRTGymnema sylvestre 280Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Trp Thr1 5
10 15Ser Met Gln Gly Glu Thr Leu Trp Arg Thr
Asp Arg Leu Ala Thr Thr 20 25
30Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5028150PRTGymnema
sylvestre 281Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val
Gly1 5 10 15Thr Thr Ile
Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20
25 30His Lys Leu Ser Gln Tyr Ser Arg Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5028250PRTGymnema sylvestre 282Met Gly Arg Gly Ser His His
His His His Leu Ala Arg Ser Val Gly1 5 10
15Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg
Asn Val Tyr 20 25 30His Lys
Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5028351PRTGymnema sylvestre
283Met Gly Arg Gly Ser His His His His His His His Ala Arg Ser Trp1
5 10 15Thr Ser Met Gln Gly Glu
Thr Leu Trp Arg Thr Asp Arg Leu Ala Ala 20 25
30Thr Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala
Pro Lys Ala 35 40 45Ser Ala Ile
5028450PRTGymnema sylvestre 284Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Leu Ser1 5 10
15Ala Leu Arg Arg Thr Glu Arg Thr Trp Asn Thr Ile His Gln Gly His
20 25 30His Leu Glu Trp Tyr Pro
Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5028550PRTGymnema sylvestre 285Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Leu Ser1 5
10 15Arg Leu Gln Thr Val Leu Asp Glu Pro Asp
Arg Ser Leu Gln Thr Arg 20 25
30Thr Asn Arg Pro His Arg Met Ile Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5028650PRTGymnema
sylvestre 286Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Val
Gly1 5 10 15Thr Thr Ile
Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr 20
25 30His Lys Leu Ser Gln Tyr Ser Arg Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5028749PRTGymnema sylvestre 287Met Gly Arg Gly Ser His His
His His His Ala Arg Ser Leu Ser Ala1 5 10
15Leu Arg Arg Thr Glu Arg Thr Trp Asn Thr Ile His Gln
Gly His His 20 25 30Leu Glu
Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser Ala 35
40 45Ile28850PRTGymnema sylvestre 288Met Gly
Arg Gly Ser His His His His His His Ala Arg Ser Cys Leu1 5
10 15Ala Thr Arg Asn Gly Phe Val Gln
Met Asn Thr Asp Arg Gly Thr Tyr 20 25
30Val Lys Arg Pro Thr Val Leu Gln Asp Ala Asn Ala Pro Lys Ala
Ser 35 40 45Ala Ile
5028950PRTGymnema sylvestreMISC_FEATURE(37)..(37)Xaa is any amino acid
289Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Lys Val1
5 10 15Asn Pro Met Arg Glu Val
Arg Cys Asn Ala Arg Cys Ile Arg Lys His 20 25
30Arg Phe Arg Leu Xaa Ile Arg Asp Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5029050PRTGymnema sylvestre 290Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Thr Met1 5 10
15Asn Thr Asn Arg Met Asp Ile Gln Arg Leu Met Thr Asn His Val Lys
20 25 30Arg Asp Ser Ser Pro Gly
Ser Ile Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5029150PRTGymnema sylvestre 291Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Met Leu1 5
10 15Leu Leu Asn Glu Thr Tyr Arg Arg Tyr Arg
Ser Trp Asp Glu Tyr Arg 20 25
30Asn Asp Ile Gly Ser Asn Leu Asp Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5029250PRTGymnema
sylvestre 292Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Gly
His1 5 10 15Arg Glu Ser
Asn Arg Val Asn Ser Asn Tyr Ala Asp Gln Leu His Ser 20
25 30Thr Pro Ile Leu Asn Thr Trp Asn Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5029350PRTGymnema sylvestre 293Met Gly Arg Gly Ser His His
His His His His Ala Arg Ser Ser Gly1 5 10
15Gln Ile Pro Tyr Lys Tyr Gly Asp Ala Ile Pro Ser Met
Leu Thr His 20 25 30Asn Ala
Glu Asn Gln Pro His Asp Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5029450PRTGymnema sylvestre
294Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Thr Pro1
5 10 15Arg Leu Arg Lys Val Tyr
Asp Leu Thr Val Thr Thr Thr Ser Ser Gln 20 25
30Ile Asp Lys Leu Gln Pro Ser Arg Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5029550PRTGymnema sylvestre 295Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Glu Gly1 5 10
15Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr
20 25 30His Lys Leu Ser Gln Tyr
Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5029650PRTGymnema sylvestre 296Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Met Arg1 5
10 15Pro Ile Leu Val Val Lys Tyr Pro Pro Tyr
Leu Gln Thr Leu Asp Asn 20 25
30Lys Arg Asp Ile Arg Gln Met Asp Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5029750PRTGymnema
sylvestre 297Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Lys
Asn1 5 10 15Asn Thr Lys
His Tyr Thr Val Val Thr Trp Cys Tyr Leu Glu Arg Lys 20
25 30Asn Gln Asn Leu Thr Ser His Thr Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5029850PRTGymnema sylvestre 298Met Gly Arg Gly Ser His His
His His His His Ala Arg Ser Ile Leu1 5 10
15Arg Ser Ala Ser Cys Ser Ala Leu Thr Asp His Lys Arg
Val Ala Tyr 20 25 30Ala Cys
Thr His Thr Glu Tyr Lys Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5029950PRTGymnema sylvestre
299Met Gly Arg Asp Ser His His His His His His Ala Arg Ser Ile Ala1
5 10 15Asn Met Tyr Gln Leu Trp
Ser Met Asn Arg Ser Asp His Asn Leu Val 20 25
30Ile Lys Lys Gln Met Ser Leu Leu Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5030050PRTGymnema sylvestre 300Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Met Leu1 5 10
15Leu Leu Asn Glu Thr Tyr Arg Arg Tyr Arg Ser Trp Asn Glu Tyr Arg
20 25 30Asn Asp Ile His Ser Asn
Leu Asp Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5030150PRTGymnema sylvestre 301Met Gly Arg Gly
Ser His His His His His His Thr Arg Ser Glu Glu1 5
10 15Asn Arg Gln Trp Arg Asn Glu Gly Ser Thr
Pro Phe Ser Ser Leu Ile 20 25
30Ser Asp Met Ser Lys Pro Ile Val Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5030250PRTGymnema
sylvestre 302Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu
Val1 5 10 15Thr Arg Leu
Leu Arg Thr His Arg Glu Glu Lys Val Phe Glu Pro Ser 20
25 30Pro Thr Gly Pro Ser Glu Lys His Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5030349PRTGymnema sylvestre 303Met Gly Arg Gly Ser His His
His His His Ala Arg Ser Asp Met Asp1 5 10
15Leu Trp Asp Leu Pro Ala Leu Ala Pro Gln Ser Thr Thr
Met Gln Met 20 25 30His Ser
Phe Thr His Met Lys Asp Ala Asn Ala Pro Lys Ala Ser Ala 35
40 45Ile30450PRTGymnema sylvestre 304Met Gly
Arg Gly Ser His His His His His His Ala Arg Ser Arg Arg1 5
10 15Val Thr Thr Glu Gly Gly Pro Lys
Trp Ile Pro Gly His His Met Arg 20 25
30Asp Asn Ile Pro Glu Ile Ala Asn Asp Ala Asn Ala Pro Lys Ala
Ser 35 40 45Ala Ile
5030548PRTGymnema sylvestre 305Met Gly Arg Gly Ser His His His His His
Ala Arg Ser Thr Trp Asn1 5 10
15Gly Arg Pro Leu His His Leu Asp His Gln Trp Tyr Pro Asp Glu Ala
20 25 30Arg Leu His Ala Ile His
Asn Ala Asn Ala Pro Lys Ala Ser Ala Ile 35 40
4530650PRTGymnema sylvestre 306Met Gly Arg Gly Ser His His
His His His His Ala Arg Ser Thr Asn1 5 10
15Arg Gly Val Asn His Thr Gly Gln Met Arg Thr Met Pro
Pro Ala Pro 20 25 30Thr Val
Glu Arg Ala Leu Asn Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5030745PRTGymnema sylvestre
307Thr Gly Arg Gly Ser His His His His His His Ala Arg Ser Pro Leu1
5 10 15Glu Leu Tyr Val Ile Thr
Arg Asp Ala Arg Thr Asp Thr Gly Pro Ser 20 25
30Ser Leu Arg Asp Ala Asn Ala Pro Lys Ala Ser Ala Ile
35 40 4530850PRTGymnema sylvestre
308Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Val1
5 10 15Ile Pro Leu Asn Glu Val
Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25
30Arg Pro Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5030949PRTGymnema sylvestre 309Met Gly Arg Gly Ser His His His His His
Ala Arg Ser Asn Val Ile1 5 10
15Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His Arg
20 25 30Ser Ser Leu Ser Ile Asp
Asp Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40
45Ile31050PRTGymnema sylvestre 310Met Gly Arg Gly Ser His
His His His His His Ala Arg Ser Val Gly1 5
10 15Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr
Arg Asn Val Tyr 20 25 30His
Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5031149PRTGymnema sylvestre
311Met Gly Arg Gly Ser His His His His His Ala Arg Ser Val Gly Thr1
5 10 15Thr Ile Arg Ile Ala Gln
Asp Thr Glu His Tyr Arg Asn Val Tyr His 20 25
30Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys
Ala Ser Ala 35 40
45Ile31249PRTGymnema sylvestre 312Met Gly Arg Gly Ser His His His His His
Ala Arg Ser Val Gly Thr1 5 10
15Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr His
20 25 30Lys Leu Ser His Tyr Ser
Arg Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40
45Ile31350PRTGymnema sylvestre 313Met Gly Arg Gly Ser His
His His His His His Ala Arg Ser Trp Thr1 5
10 15Ser Met Gln Gly Glu Thr Leu Trp Arg Thr Asp Arg
Leu Ala Thr Thr 20 25 30Lys
Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5031449PRTGymnema sylvestre
314Met Gly Arg Gly Ser His His His His His Ala Arg Ser Pro Leu Trp1
5 10 15Tyr His Tyr Asn Cys Trp
Asp Thr Ile Cys Leu Ala Asp Trp Leu Lys 20 25
30Asp Arg Pro His Gly Val Tyr Asp Ala Asn Ala Pro Lys
Ala Ser Ala 35 40
45Ile31550PRTGymnema sylvestre 315Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Trp Thr1 5 10
15Ser Met Gln Gly Glu Thr Leu Trp Arg Thr Asp Arg Leu Ala Thr Thr
20 25 30Lys Thr Ser Met Ser His
Pro Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5031650PRTGymnema sylvestre 316Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Leu Ser1 5
10 15Ala Leu Met Arg Thr Glu Arg Thr Trp Asn
Thr Ile His Gln Gly His 20 25
30His Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5031749PRTGymnema
sylvestre 317Met Gly Arg Gly Ser His His His His His Ala Arg Ser Leu Ser
Ala1 5 10 15Leu Met Arg
Thr Glu Arg Thr Trp Asn Thr Ile His Gln Gly His His 20
25 30Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn
Ala Pro Lys Ala Ser Ala 35 40
45Ile31849PRTGymnema sylvestre 318Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Cys Leu1 5 10
15Ala Thr Arg Asn Gly Phe Val Met Asn Thr Asp Arg Gly Thr Tyr Val
20 25 30Lys Arg Pro Thr Val Leu
Gln Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40
45Ile31950PRTGymnema sylvestre 319Met Gly Arg Gly Ser His
His His His His His Ala Arg Ser Thr Met1 5
10 15Asn Thr Asn Arg Met Asp Ile Gln Arg Leu Met Thr
Asn His Val Lys 20 25 30Arg
Asp Ser Ser Pro Gly Ser Ile Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5032050PRTGymnema sylvestre
320Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ser Phe1
5 10 15Asn Lys Val Gly Arg Val
Asp Ser Glu Phe Gly Thr Lys Ala Asn Ser 20 25
30His Gln Ile Pro Ser Gly Glu Leu Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5032150PRTGymnema sylvestre 321Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Ile Lys1 5 10
15Tyr Trp Met Ile Pro Ser Trp Asn Leu Tyr Pro Trp Leu Leu Met Tyr
20 25 30Asp Thr Leu Ile His Pro
Thr Met Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5032250PRTGymnema sylvestre 322Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Trp Trp1 5
10 15Thr Arg Met Gln Ile Pro Thr Ser Trp Tyr
Trp Tyr Thr Tyr Trp Ile 20 25
30Asn His Leu Gln Lys His Asp Ile Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5032350PRTGymnema
sylvestre 323Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp
Arg1 5 10 15Trp His Asn
Trp Gly Leu Ser Asp Thr Val Ala Ser His Pro Asp Ala 20
25 30Ser Asn Ser Leu Asn Met Met Tyr Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5032450PRTGymnema sylvestre 324Met Gly Arg Gly Ser His His
His His His Asp Ala Arg Ser Ser His1 5 10
15Trp Ser Asn Ala Asp His Ile Gly Pro Ser Arg Cys Leu
Gly Cys Thr 20 25 30Met Thr
Thr Leu Ile Arg Leu Pro Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5032550PRTGymnema sylvestre
325Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Arg Ser1
5 10 15Ile Pro Val Arg Ile Gln
Gly Asn Pro Gly Asn Ser His Tyr Arg Leu 20 25
30Met Gly Ala Ser Met Val His Gly Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5032650PRTGymnema sylvestre 326Met Gly Arg Asp Ser His His His His His
His Ala Arg Ser Ile Ala1 5 10
15Asn Met Tyr Gln Leu Trp Ser Met Asn Arg Ser Asp His Asn Leu Val
20 25 30Ile Lys Lys Gln Met Ser
Leu Leu Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5032748PRTGymnema sylvestre 327Met Gly Arg Ser
His His His His His Ala Arg Ser Gly Lys Phe Arg1 5
10 15His Glu Ile Tyr Asn Met Glu Trp Pro Leu
Ala Leu Glu Arg Tyr Trp 20 25
30Asp Tyr His Gly Glu Pro Asp Ala Asn Ala Pro Lys Ala Ser Ala Ile
35 40 4532850PRTGymnema sylvestre
328Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Leu Glu1
5 10 15Thr Thr Thr Thr Ser Leu
Met Asn Glu Glu Asp Ala Trp Asn Trp Thr20 25
30Ile Glu Lys Ser Arg His Ile Glu Asp Ala Asn Ala Pro Lys Ala Ser35
40 45Ala Ile5032950PRTGymnema sylvestre
329Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ile Met1
5 10 15Tyr Met His Trp Gln Trp
Ala Val Asn Arg Met Gly His Ala Thr Ala 20 25
30Met Ser Thr Leu Ala Asn Ala Tyr Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5033049PRTGymnema sylvestre 330Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Asn Asp1 5 10
15Ile Pro Leu Asn Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His Arg
20 25 30Ser Arg Leu Thr Ile Asp
Asp Asp Ala Asn Ala Pro Lys Ala Ser Ala 35 40
45Ile33149PRTGymnema sylvestre 331Met Gly Arg Gly Ser His
His His His His His Ala Arg Ser Asn Val1 5
10 15Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp
Asp Arg Pro His 20 25 30Arg
Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala33249PRTGymnema sylvestre 332Met
Gly Arg Gly Ser His His His His His Arg Ala Arg Ser Asn Val1
5 10 15Ile Pro Leu Asn Glu Val Trp
Tyr Asp Thr Gly Trp Asp Arg Pro His 20 25
30Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys
Ala Ser 35 40
45Ala33349PRTGymnema sylvestre 333Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Val Gly1 5 10
15Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Tyr Arg Asn Val Tyr
20 25 30His Lys Leu Ser Gln Tyr
Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala33448PRTGymnema sylvestre 334Met Gly Arg Gly Ser His
His His Gln His Asn Ala Arg Ser Val Ala1 5
10 15Thr Thr Ile Pro Asp Arg Pro Gly His Gly Thr Leu
Pro Glu Arg Leu 20 25 30Pro
Gln Ala Leu Pro Glu Leu Pro Gly Arg Arg Ser Glu Gly Ile Arg 35
40 4533549PRTGymnema sylvestre 335Met Gly
Arg Gly Ser His His His His His Ala Arg Ser Val Gly Thr1 5
10 15Thr Ile Arg Ile Ala Gln Asp Thr
Glu His Tyr Arg Asn Val Tyr His 20 25
30Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser
Ala 35 40 45Ile33649PRTGymnema
sylvestre 336Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Trp
Thr1 5 10 15Ser Met Gln
Gly Glu Thr Leu Trp Arg Thr Asp Arg Leu Ala Thr Thr 20
25 30Lys Thr Ser Met Ser His Pro Pro Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala33749PRTGymnema sylvestre 337Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Trp Gln1 5 10
15Pro Glu Val Lys Met Ser Ser Leu Val Asp Thr Ser Gln Thr Val Gly
20 25 30Ala Ala Val Glu Thr Arg
Thr Thr Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala33849PRTGymnema sylvestre 338Met Gly Arg Gly Ser His
His His His His His Ala Arg Ser Leu Ser1 5
10 15Ala Leu Arg Arg Thr Glu Arg Thr Trp Asn Thr Ile
His Gln Gly His 20 25 30His
Leu Glu Trp Tyr Pro Pro Ala Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala33949PRTGymnema sylvestre 339Met
Gly Arg Gly Ser His His His His His His Ala Arg Ser Cys Leu1
5 10 15Ala Arg Thr Asn Gly Phe Val
Gln Met Asn Thr Asp Arg Gly Thr Tyr 20 25
30Val Lys Arg Pro Thr Val Leu Gln Asp Ala Asn Ala Pro Lys
Ala Ser 35 40
45Ala34049PRTGymnema sylvestre 340Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Thr Met1 5 10
15Asn Thr Asn Arg Met Asp Ile Gln Arg Leu Met Thr Asn His Val Lys
20 25 30Arg Asp Ser Ser Pro Gly
Ser Ile Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala34149PRTGymnema sylvestre 341Met Gly Arg Gly Ser His
His His His His His Ala Arg Ser Gln Val1 5
10 15Thr Trp His His Leu Ala Asp Thr Val Thr Thr Lys
Asn Arg Lys Cys 20 25 30Thr
Asp Ser Tyr Ile Gly Trp Asn Glu Leu Thr Leu Arg Arg His Pro 35
40 45Leu34249PRTGymnema sylvestre 342Met
Gly Arg Gly Ser His His His His His His Ala Arg Ser Thr Gly1
5 10 15Gly Pro Thr Gly Thr Ser Ala
Ser Ala Gly Pro Thr Ser Ala Thr Arg 20 25
30Ser Pro Pro Gly Gly Pro Arg Arg Thr Leu Thr Leu Arg Arg
His Pro 35 40
45Leu34343PRTGymnema sylvestre 343Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Gly Lys1 5 10
15Val Arg Gly His Thr Lys Glu Thr Pro Pro Thr Glu Phe Gly Leu Ser
20 25 30Leu Met Asp Ala Asn Ala
Pro Lys Ala Ser Ala 35 4034449PRTGymnema
sylvestre 344Met Gly Arg Gly Ser His His His His His His Leu Asp Leu Trp
Gly1 5 10 15Pro Pro Ser
Gly Ser Pro Arg Thr Arg Ser Thr Thr Gly Thr Ser Thr 20
25 30Thr Ser Ser Pro Ser Thr Pro Gly Thr Leu
Thr Leu Arg Arg His Pro 35 40
45His34549PRTGymnema sylvestre 345Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Pro Thr1 5 10
15Met Arg Arg His Ile Arg Arg Ala Leu Tyr Pro Tyr Ser Thr Arg Arg
20 25 30Ser Leu Leu Thr Ser Ala
Pro Val Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala34649PRTGymnema sylvestre 346Met Gly Arg Gly Ser His
His His His His His Ala Arg Ser Ser Val1 5
10 15His Trp Ser Tyr Cys Gly Ala Glu Val Lys Lys Asp
Trp Tyr Gln His 20 25 30Thr
Ala Trp Thr Lys Asn His Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala34749PRTGymnema sylvestre 347Met
Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn Met1
5 10 15Asn Thr Arg Arg Met Asp Ile
Arg Asn Leu Ile Thr Lys Arg Val Lys 20 25
30Lys Asp Tyr Ser Pro Gly Ser Lys Asp Ala Asn Ala Pro Lys
Ala Ser 35 40
45Ala34849PRTGymnema sylvestre 348Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Val Asp1 5 10
15Asp Thr Gly His Leu Leu His Thr Gly Arg Leu Met Arg Thr Pro Ser
20 25 30Thr Asn Ser Trp His Thr
Leu Asn Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala34949PRTGymnema sylvestre 349Met Gly Arg Gly Ser His
His His His His His Ala Arg Ser Ser Leu1 5
10 15Asn Lys Val Gly Arg Val Asp Ser Glu Phe Gly Thr
Lys Ala Asn Ser 20 25 30His
Gln Ile Pro Ser Gly Glu Leu Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala35049PRTGymnema sylvestre 350Met
Gly Arg Gly Ser His His His His His His Ala Arg Ser His Ser1
5 10 15Arg His Glu Trp Thr Ser Thr
Pro Arg Arg Arg Arg Ser Thr Gly Pro 20 25
30Gly Ser Arg Trp Ala Ser Gly Thr Asp Ala Asn Ala Pro Lys
Ala Ser 35 40
45Ala35149PRTGymnema sylvestre 351Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Gly Arg1 5 10
15Tyr His Arg Asp Arg Trp Leu Ala Thr Met Arg Tyr Pro Asp Pro Ser
20 25 30Gln Val Trp Ser Arg Tyr
Val Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala35249PRTGymnema sylvestre 352Met Gly Arg Gly Ser His
His His His His His Ala Arg Ser Trp Arg1 5
10 15Trp His Asn Trp Gly Leu Ser Asp Thr Val Ala Ser
His Pro Asp Ala 20 25 30Ser
Asn Ser Leu Asn Met Met Tyr Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala35349PRTGymnema sylvestre 353Met
Gly Arg Gly Ser His His His His His His Ala Arg Ser Pro Leu1
5 10 15Trp Tyr His Tyr Asn Cys Trp
Asp Thr Ile Cys Leu Ala Asp Trp Leu 20 25
30Lys Asp Arg Pro His Gly Val Tyr Asp Ala Asn Ala Pro Lys
Ala Ser 35 40
45Ala35448PRTGymnema sylvestre 354Met Gly Arg Gly Ser His His His His Ala
Arg Ser Asn Val Ile Pro1 5 10
15Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His Arg Ser
20 25 30Arg Leu Ser Ile Asp Asp
Asp Ala Asn Ala Pro Lys Ala Ser Ala Ile 35 40
4535547PRTGymnema sylvestre 355Met Gly Leu Leu His His His
His Ala Arg Ser Asn Val Ile Pro Leu1 5 10
15Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His
Arg Ser Arg 20 25 30Leu Ser
Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser Ala Ile 35
40 4535650PRTGymnema sylvestre 356Met Gly Arg Ser
Ser His His His His His His Ala Arg Ser Asn Val1 5
10 15Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr
Gly Trp Asp Arg Pro His 20 25
30Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5035750PRTGymnema
sylvestre 357Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn
Val1 5 10 15Ile Pro Leu
Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20
25 30Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5035849PRTGymnema sylvestre 358Met Gly Arg Gly Ser His His
His His His His Ala Arg Ser Asn Val1 5 10
15Ile Pro Leu Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp
Arg Pro His 20 25 30Arg Ser
Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Asn35948PRTGymnema sylvestre 359Met Gly
Arg Ser His His His His His His Ala Arg Ser Asn Val Ile1 5
10 15Pro Leu Asn Glu Val Trp Tyr Asp
Thr Gly Trp Asp Arg Pro His Arg 20 25
30Ser Arg Leu Ser Ile Asp Asp Asp Ala Asn Ala Pro Lys Ala Ser
Ala 35 40 4536046PRTGymnema
sylvestre 360Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn
Val1 5 10 15Ile Pro Leu
Asn Glu Val Trp Tyr Asp Thr Gly Trp Asp Arg Pro His 20
25 30Arg Ser Arg Leu Ser Ile Asp Asp Asp Ala
Asn Ala Pro Arg 35 40
4536150PRTGymnema sylvestre 361Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Val Gly1 5 10
15Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Thr Arg Asn Val Tyr
20 25 30His Lys Leu Ser Gln Tyr
Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5036244PRTGymnema sylvestre 362Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Val Gly1 5
10 15Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu
His Thr Arg Asn Val Tyr 20 25
30His Lys Leu Ser Gln Tyr Ser Arg Asp Ala Asn Ala 35
4036350PRTGymnema sylvestre 363Met Gly Arg Gly Ser His His His His
His His Ala Arg Ser Val Gly1 5 10
15Thr Thr Ile Arg Ile Ala Gln Asp Thr Glu His Thr Arg Asn Val
Tyr 20 25 30His Lys Leu Ser
Gln Tyr Ser Arg Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5036450PRTGymnema sylvestre 364Met Gly
Arg Gly Ser His His His His His His Ala Arg Ser Trp Thr1 5
10 15Ser Met Gln Gly Glu Thr Leu Trp
Arg Thr Asp Arg Leu Ala Thr Thr 20 25
30Lys Thr Ser Met Ser His Pro Pro Asp Ala Asn Ala Pro Lys Ala
Ser 35 40 45Ala Ile
5036550PRTGymnema sylvestre 365Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Cys Leu1 5 10
15Ala Thr Arg Asn Gly Phe Glu Gln Met Asn Thr Asp Arg Gly Thr Tyr
20 25 30Val Lys Arg Thr Thr Val
Leu Gln Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5036650PRTGymnema sylvestre 366Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Trp Arg1 5
10 15Asp Thr Arg Lys Leu His Met Arg His Tyr
Phe Pro Leu Ala Ile Asp 20 25
30Ser Tyr Trp Asp His Thr Leu Arg Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5036750PRTGymnema
sylvestre 367Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Ser
Pro1 5 10 15Leu Trp Tyr
His Tyr Asn Cys Trp Asp Thr Ile Cys Leu Ala Asp Trp 20
25 30Leu Lys Asp Arg Pro His Gly Val Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5036851PRTGymnema sylvestre 368Met Gly Arg Gly Ser His His
His His His His His Ala Arg Ser Pro1 5 10
15Leu Trp Tyr His Tyr Asn Cys Trp Asp Thr Ile Cys Leu
Ala Asp Trp 20 25 30Leu Lys
Asp Arg Pro His Gly Val Tyr Asp Ala Asn Ala Pro Lys Ala 35
40 45Ser Ala Ile 5036950PRTGymnema sylvestre
369Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Pro Leu1
5 10 15Trp Tyr His Tyr Asn Cys
Trp Asp Thr Ile Cys Leu Ala Asp Trp Leu 20 25
30Lys Asp Arg Pro His Gly Val Tyr Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
5037050PRTGymnema sylvestre 370Met Gly Arg Gly Ser His His His His His
His Ala Arg Ser Gly Arg1 5 10
15Tyr His Arg Asp Arg Trp Leu Ala Thr Met Arg Tyr Pro Asp Pro Ser
20 25 30Gln Val Trp Ser Arg Tyr
Val Pro Asp Ala Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5037150PRTGymnema sylvestre 371Met Gly Arg Gly
Ser His His His His His His Ala Arg Ser Thr Met1 5
10 15Asn Thr Asn Arg Met Asp Ile Gln Arg Leu
Met Thr Asn His Val Lys 20 25
30Arg Asp Ser Ser Pro Gly Ser Ile Asp Ala Asn Ala Pro Lys Ala Ser
35 40 45Ala Ile 5037250PRTGymnema
sylvestre 372Met Gly Arg Gly Ser His His His His His His Ala Arg Ser Asn
Leu1 5 10 15Tyr Ile Thr
Gly Glu Phe Lys Arg Gln Thr Asp Asn Asn Gly Ser Glu 20
25 30Leu Arg Arg Met Ser Arg Pro Arg Asp Ala
Asn Ala Pro Lys Ala Ser 35 40
45Ala Ile 5037350PRTGymnema sylvestre 373Met Gly Arg Gly Ser His His
His His His His Ala Arg Ser Asn Cys1 5 10
15Leu Ile Ser Leu Thr Ala Glu Glu Lys Ala Leu Asn Arg
Met Met Asn 20 25 30Val Ser
Val Pro Arg Val Met Thr Asp Ala Asn Ala Pro Lys Ala Ser 35
40 45Ala Ile 5037450PRTGymnema sylvestre
374Met Gly Arg Asp Ser His His His His His His Ala Arg Ser Ile Ala1
5 10 15Asn Met Tyr Gln Leu Trp
Ser Met Asn Arg Ser Asp His Asn Leu Val 20 25
30Ile Lys Lys Gln Met Ser Leu Leu Asp Ala Asn Ala Pro
Lys Ala Ser 35 40 45Ala Ile
50
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