Composition comprising recombinant clostridium neurotoxin

Abstract

The invention provides methods for producing soluble di-chain BoNT/A protein.

Claims

1. A liquid pharmaceutical composition comprising: an active di-chain BoNT/A protein; Lys-C; a non-protein stabilizing agent that is a surfactant; and water; wherein: the composition does not comprise a protein stabilizing agent; the Lys-C is present at a concentration of less than 400 pg Lys-C per 100 ng BoNT/A protein; and less than 2% of the BoNT/A in the composition is single-chain BoNT/A.

2. The liquid pharmaceutical composition of claim 1, wherein the composition contains Lys-C at a concentration of less than 300 pg Lys-C per 100 ng BoNT/A protein.

3. The liquid pharmaceutical composition of claim 1, further comprising: sodium chloride; a buffer to maintain pH between 5.5 and 7.5; and a disaccharide; wherein the water is sterile water.

4. The liquid pharmaceutical composition of claim 2, wherein the composition contains Lys-C at a concentration of less than 200 pg Lys-C per 100 ng BoNT/A protein.

5. The liquid pharmaceutical composition of claim 2, wherein the composition contains Lys-C at a concentration of less than 100 pg Lys-C per 100 ng BoNT/A protein.

6. The liquid pharmaceutical composition of claim 2, wherein the composition contains Lys-C at a concentration of less than 50 pg Lys-C per 100 ng BoNT/A protein.

7. The liquid pharmaceutical composition of claim 2, wherein the composition contains Lys-C at a concentration of less than 20 pg Lys-C per 100 ng BoNT/A protein.

8. The liquid pharmaceutical composition of claim 2, wherein the composition contains Lys-C at a concentration of less than 10 pg Lys-C per 100 ng BoNT/A protein.

9. The liquid pharmaceutical composition of claim 1, wherein less than 1% of the BoNT/A in the composition is single-chain BoNT/A.

10. The liquid pharmaceutical composition of claim 1, produced using a method wherein a soluble single-chain BoNT/A protein is contacted with Lys-C in solution and, following such contact, the BoNT/A is separated from Lys-C by contacting the solution containing the BoNT/A and Lys-C with a hydrophobic surface, wherein the BoNT/A binds with preference to the hydrophobic surface.

11. The liquid pharmaceutical composition of claim 3 consisting of: BoNT/A protein wherein less than 2% of the protein is in single-chain form; a non-protein stabilizing agent that is a surfactant; water; Lys-C at less than 400 pg Lys-C per 100 ng BoNT/A protein; sodium chloride; a buffer to maintain pH between 5.5 and 7.5; and a disaccharide.

Description

LIST OF FIGURES

(1) FIG. 1: Elution profiles from Phenyl High Performance (PhHP) column on which the separation of Lys-C (A.sub.405-bars) from BoNT/A was assessed (A.sub.280-dotted line).

(2) FIG. 2: Elution profiles from Phenyl Fast Flow High substitution (PhFF-Hi) column on which the separation of Lys-C (A.sub.405-bars) from BoNT/A was assessed (A.sub.280-dotted line).

(3) FIG. 3: Elution profiles from Butyl High Performance (BuHP) column on which the separation of Lys-C (A.sub.405-bars) from BoNT/A was assessed (A.sub.280-dotted line).

(4) FIG. 4: Elution profiles from Sulphopropyl High Performance (SPHP) column on which the separation of Lys-C (A.sub.405-bars) from BoNT/A was assessed (A.sub.280-dotted line).

(5) FIG. 5: Elution profiles from Sulphopropyl Fast Flow (SPFF) column on which the separation of Lys-C (A.sub.405-bars) from BoNT/A was assessed (A.sub.280-dotted line).

(6) FIG. 6: Elution profiles from Carboxylmethyl Fast Flow (CMFF) column on which the separation of Lys-C (A.sub.405-bars) from BoNT/A was assessed (A.sub.280-dotted line).

(7) FIG. 7: Elution profiles from Quaternary amine High Performance (QHP) column on which the separation of Lys-C (A.sub.405-bars) from BoNT/A was assessed (A.sub.280-dotted line).

(8) FIG. 8: Elution profiles from Quaternary amine Fast Flow (QFF) column on which the separation of Lys-C (A.sub.405-bars) from BoNT/A was assessed (A.sub.280-dotted line).

(9) FIG. 9: Elution profiles from Diethylaminopropyl (DEAP, “ANX”) column on which the separation of Lys-C (A.sub.405-bars) from BoNT/A was assessed (A.sub.280-dotted line).

(10) FIG. 10: Elution profiles from Diethylaminoethyl (DEAE) column on which the separation of Lys-C (A.sub.405-bars) from BoNT/A was assessed (A.sub.280-dotted line).

(11) FIG. 11: Removal of Lys-C from rBoNT/A1 by Phenyl HP HIC. Fractions taken from the PhHP HIC polish step, were analysed by SDS-PAGE (top). Recombinant BoNT/A1 has a molecular weight of ˜149 kiloDaltons (kDa) and molecular weight markers (Benchmark) are labelled in kDa. Samples of the same fractions were also tested in the colorimetric Lys-C assay (bottom) where the cleavage of substrate releases a yellow chromophore (in the black box).

(12) FIG. 12: Removal of Lys-C from rBoNT/A2(0) by Phenyl HP HIC. Fractions taken from the Phenyl HP HIC polish step, were analysed by SDS-PAGE (top). Recombinant BoNT/A1 has a molecular weight of ˜149 kDa and molecular weight markers (Benchmark) are labelled in kDa. Samples of the same fractions were also tested in the colorimetric Lys-C assay (bottom) where the cleavage of substrate releases a yellow chromophore (in the black box).

(13) FIG. 13: Removal of Lys-C from rBoNT/A5(0) by Phenyl HP HIC. Fractions taken from the Phenyl HP HIC polish step, were analysed by SDS-PAGE (top). Recombinant BoNT/A5(0) has a molecular weight of ˜149 kDa and molecular weight markers (Benchmark) are labelled in kDa. Samples of the same fractions were also tested in the colorimetric Lys-C assay (bottom) where the cleavage of substrate releases a yellow chromophore (in the black box).

(14) FIG. 14: Removal of Lys-C from rBoNT/A6(0) by Phenyl HP HIC. Fractions taken from the Phenyl HP HIC polish step, were analysed by SDS-PAGE (top). Recombinant BoNT/A6(0) has a molecular weight of ˜149 kDa and molecular weight markers (Benchmark) are labelled in kDa. Samples of the same fractions were also tested in the colorimetric Lys-C assay (bottom) where the cleavage of substrate releases a yellow chromophore (in the black box).

EXAMPLES

Example 1—Culturing of Host and Expression of Soluble rBoNT/A Protein

(15) A single colony of BoNT/A transformed in BLR (DE3) cells is used to inoculate a 250 mL conical flask containing 100 mL modified Terrific Broth (mTB) supplemented with 0.2% glucosamine and 30 μg/mL kanamycin. This method would be equally applicable when using a Microbank bead or glycerol stock (10-100 μL) to inoculate the flask.

(16) The flask is incubated for 16 hours at 37° C. with 250 RPM shaking. 10 mL of this starter culture is used to inoculate 2 L conical flasks each containing 1 L supplemented with 0.2% glucosamine and 30 μg/mL kanamycin. Cells are grown at 37° C. for 2 hours at 225 RPM until an OD.sub.600 of 0.5 is reached. At this point, the culture temperature is dropped to 16° C. After 1 hour, the cells are induced to express BoNT/A by addition of 1 mM IPTG for 20 hours. Cells are harvested by centrifugation for 20 min at 4° C., weighed and then stored at −20° C.

Example 2—Extraction of BoNT/A Protein from Host and Analysis of Expression Level

(17) Expression cell pastes of rBoNT/A are thawed at room temperature and resuspended by pipetting in 3 mL of Tris-NaCl re-suspension buffer per gram of cells supplemented with 10 μL benzonase. Cells are lysed by sonication at 100 W−10×30 s on+45 s off. The lysate is centrifuged at 4000×g for 1 h at 4° C. to obtain the soluble rBoNT/A in the supernatant.

(18) Bradford Assay to Determine Total Protein Concentration of Prepared Lysates

(19) A sample (50 μL) of either diluted rBoNT/A lysate or BSA standard is added to 1 mL disposable cuvettes. 450 μL of Coomassie Bradford Assay reagent is added to each cuvette and allowed to incubate at room temperature for 10 minutes before reading A.sub.600. The values obtained for the BSA standards are used to determine the amount of protein in the lysate samples.

(20) Semi-Quantitative Western Blot Analysis

(21) A commercial sample of BoNT/A protein purchased from Metabiologics is used to make up SDS-PAGE standards. SDS-PAGE samples of the lysate samples from the expressed cell cultures are then prepared to a known total protein concentration. These samples are loaded onto a polyacrylamide gel and run at 200 V for 50 minutes. Protein bands are electroblotted onto nitrocellulose membrane in methanol free blotting buffer at 0.4 mA for 1 hour. The membranes are blocked for 1 hour with 0.5% BSA in PBS-0.1% Tween 20 and then probed with an antibody to BoNT/A for 1 hour. The blots are further probed with HRP conjugated secondary antibody, developed with SuperSignal DuraWest substrate, and imaged using a Syngene Imaging Instrument.

Example 3—Activation of Botulinum Neurotoxin A (BoNT/A) by Lys-C

(22) Single chain recombinant BoNT/A1 (0.5 mg/mL) dissolved in buffer (50 mM Tris/HCl pH 8.0, 125 mM NaCl) was proteolytically activated by Lys-C (at 1:500 to 1:2500 enzyme:substrate ratio) at 37° C. or 4° C., over a period of 2-20 hr, before the reaction was inhibited with 0.4 μM AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), a specific serine protease inhibitor. This yields the mature di-chain form of BoNT/A1, where the heavy chain is linked to the light chain by a single disulphide bond (data not shown).

(23) The cleavage site was determined to be identical to the endogenous protein by N-terminal sequencing and mass spectrometry, confirming Lys-C to be the activating enzyme of choice (data not shown).

(24) Endoproteinase Lys-C cleavage tests demonstrated that Lys-C cleaved rBoNT/A1 at very low concentrations and remained active over a period of days (data not shown).

Example 4—Purification of Target BoNT/A Protein Free from Activating Protease

(25) Using the BoNT/A primary protein sequence, the properties of BoNT/A and Lys-C were investigated (see Table 2). The predicted properties suggested that both Lys-C and BoNT/A have a similar mean hydropathicity (GRAVY value), but large charge difference at pH 4.5 and 8 (see Table 2).

(26) TABLE-US-00002 TABLE 2 Predicted properties of Lys-C and BoNT/A Property (calculated) Lys-C BoNT/A pI 6.70 6.05 % residues charged (DEKR) 13 25 Grand Average of Hydropathicity −0.30 −0.37 (GRAVY)* Charge at pH 8.0 −5 −12 Charge at pH 4.5 +13 +72 (*GRAVY = the mean hydropathicity per residue of a molecule (a positive value indicates a hydrophobic molecule))

(27) Based on this information alone, it was predicted that ion exchange (IEX) chromatography would resolve Lys-C from BoNT/A. Therefore, various chromatographic means of purification, including IEX chromatography, were investigated (see below).

Example 5—Screening of Fast Protein Liquid Chromatography (FPLC) Columns for Separating Lys-C from BoNT/A

(28) FPLC Purification

(29) After BoNT/A1 activation with Lys-C, a number of FPLC columns were tested for polishing and removal of Lys-C: three hydrophobic interaction chromatography (HIC) columns: Phenyl High Performance (PhHP), Phenyl Fast Flow High substitution (PhFF-Hi), and Butyl HP (BuHP) were tested with Tris pH 8; three cation exchange chromatography (CEC) columns: Sulphopropyl High Performance (SPHP), Sulphopropyl Fast Flow (SPFF), and Carboxylmethyl Fast Flow (CMFF) were tested with sodium acetate pH 4.5; and four anion exchange chromatography (AEC) columns: Quaternary amine High Performance (QHP), Quaternary amine Fast Flow (QFF), Diethylaminopropyl (DEAP, “ANX”), and Diethylaminoethyl (DEAE)) were tested with Tris pH 8.

(30) Once a sample was loaded onto a column, the column was washed through with buffer to remove any non-specifically bound molecules before applying a linear elution gradient of increasing or decreasing concentration of salt (HIC and CEC/AEC, respectively).

(31) The reaction conditions and purification runs vary between different column types (see Table 3 below).

(32) TABLE-US-00003 TABLE 3 Screening conditions for all columns tested for Lys-C resolution BoNT/A1 Lys-C loaded loaded Column (mg) (μg) Buffer Elution gradient PhHP 1.9 1.5 High-salt, Tris pH 8 1-0M, 15 CV BuHP 1.9 1.5 High-salt, Tris pH 8 1-0M, 15 CV PhFF-Hi 1.5 1.2 High-salt, Tris pH 8 1-0M, 15 CV SPHP 3.4 3.0 Low-salt, NaOAc 0-0.5M, 30 CV pH 4.5 SPFF 3.4 3.0 Low-salt, NaOAc 0-0.5M, 30 CV pH 4.5 CMFF 3.4 3.0 Low-salt, NaOAc 0-0.5M, 30 CV pH 4.5 QHP 2.7 2.0 Low-salt, Tris pH 8 0-0.5M, 30 CV QFF 2.7 2.0 Low-salt, Tris pH 8 0-0.5M, 30 CV ANX 2.7 2.0 Low-salt, Tris pH 8 0-0.5M, 30 CV DEAE 2.7 2.0 Low-salt, Tris pH 8 0-0.5M, 30 CV

(33) FIGS. 1 to 10 show the elution profiles (A.sub.280) of the BoNT/A1 protein from the various chromatographic columns (dotted lines). The different reaction conditions and purification runs used for the different columns explains the different scales used.

(34) Fractions collected during the elution gradient were analysed with a colorimetric assay to assess Lys-C activity.

(35) Lys-C Activity Colorimetric Assay

(36) This assay involved cleaving a colourless substrate to produce a yellow chromophore that may be detected photometrically by absorption of 405 nm light (A.sub.405). Thus, this assay provided a simple method to determine if Lys-C was present in each fraction.

(37) Each elution fraction was analysed using said colorimetric Lys-C activity assay and the A.sub.405 nm measured.

(38) The amount (measured in terms of A.sub.405) of Lys-C in each of the elution fractions is shown as the bars in FIGS. 1 to 10.

(39) Results By comparing the A.sub.405 and A.sub.280 data (in the graphs of FIGS. 1 to 10), it was possible to deduce which column/s provide the best resolution of Lys-C from BoNT/A.

(40) The different alkyl/aryl groups (Bu and Ph) of the three HIC columns used provide different ligands to which various proteins may interact via the hydrophobic effect. This interaction is further influenced by the density (degree of substitution (Hi/Lo) and bead size (FF/HP)) of these hydrophobic groups. For the CEC columns, the pH of the sample is adjusted to below that of the target protein pI so that it attains an overall net positive charge and is thus able to bind to the column. The different ligands present on each type of column provide different charge densities, and the interaction with different proteins is also influenced by ligand density. These variables similarly apply to the AEC columns where the different chemical groups display different charge densities. In this instance, the pH of the sample is adjusted to above that of the target protein pI so that it attains an overall net negative charge.

(41) The graphical data from FIGS. 1 to 10 is summarised qualitatively in Table 4.

(42) TABLE-US-00004 TABLE 4 Summary of qualitative Lys-C/BoNT resolution data Lys-C Chromatography resolution* Hydrophobic PhHP ✓✓✓ interaction.sup.a BuHP ✓✓✓ PhFF-Hi ✓✓ Ion exchange.sup.b Cationic SPHP ✓ SPFF ✓ CMFF ✓ Anionic QHP ✓✓ QFF ✓ ANX ✓ DEAE ✓ *Apparent resolution of Lys-C with respect to rBoNT/A1. ✓✓✓ = Good, ✓✓ = OK, ✓ = Poor .sup.aPhenyl High Performance (PhHP), Phenyl Fast Flow High substitution (PhFF-Hi), Butyl HP (BuHP) .sup.bSulphopropyl High Performance (SPHP), Sulphopropyl Fast Flow (SPFF), Carboxylmethyl Fast Flow (CMFF), Quaternary amine High Performance (QHP), Quaternary amine Fast Flow (QFF), Diethylaminopropyl (DEAP, “ANX”), and Diethylaminoethyl (DEAE)

(43) Percentage Recoveries of Lys-C and Purification after Elution from Each Column Type

(44) The total Lys-C signal in each fraction was normalised to the mean A.sub.405 value from the last 5 fractions of the chromatographic step. From this, the percentage Lys-C present in the protein peak fractions was calculated to indicate the degree of separation of Lys-C from protein based on the elution fractions.

(45) With regard to the target protein, it is assumed that the BoNT/A molecule elutes under the major peak (i.e., 100% recovery); therefore, the degree of purification may be expressed as a percentage of the total protein loaded (Table 5). From this, it appears that CEC is not able to resolve Lys-C from BoNT/A1. The high performance AEC column, QHP, showed some ability to resolve Lys-C from BoNT/A1. However, it was significantly less effective than the two high performance HIC columns. Therefore, the results demonstrate that, comparing like-for-like (i.e. standard performance vs standard performance and high performance vs high performance), the HIC columns showed improved resolution of Lys-C from BonT/A1 than either the CEC or AEC columns.

(46) The two most promising candidates involve HIC-PhHP and BuHP. Interestingly, these columns both use high performance beads.

(47) The major difference between high performance media and others is that the average particle size is smaller (34 μm vs. 90 μm) and more uniform (24-44 μm vs. 44-165 μm). This is consistent with reported improvements in performance with analytical columns that use smaller sized beads (mean sizes between 3-30 μm) (GE Healthcare handbooks 11-0004-21 & 11-0012-69 and data files 18-1172-87 AE & 18-1172-88 AD).

(48) TABLE-US-00005 TABLE 5 Summary of column performance % LysC in protein peak Column fractions (normalised) % Purification PhHP 5 11 BuHP 7 11 PhFF-Hi 43 22 SPHP 85 20 SPFF 82 11 CMFF 81 10 QHP 26 9 QFF 51 6 ANX 70 7 DEAE 74 9

(49) PhHP HIC was chosen as the final polish step to resolve away the Lys-C from BoNT/A (see Example 6 below).

Example 6—Activation and Final Purification of Recombinant Botulinum Neurotoxin Sub-Serotype A1 (rBoNT/A1)

(50) Single chain rBoNT/A1 was purified by fast protein liquid chromatography (FPLC) using high-performance butyl sepharose hydrophobic interaction chromatography (Butyl HP HIC) for capture followed by an intermediate purification step with high-performance quaternary ammonium sepharose anionic exchange chromatography (Q HP AEC). This molecule was then incubated with 0.4 μg/mL Lys-C at 37° C. for 2 h to yield the active di-chain.

(51) The Lys-C was resolved from the activated rBoNT/A1 using high-performance phenyl sepharose hydrophobic interaction chromatography (PhHP HIC). This involved adjusting the reaction mixture with a high-salt Tris buffer before loading onto the PhHP column, followed by a high-salt wash and subsequent protein elution with a linear gradient to a low-salt Tris buffer. Elution fractions were analysed by denaturing gel electrophoresis (SDS PAGE—FIG. 11 top) and a colorimetric Lys-C activity assay (FIG. 11 bottom). Lys-C was demonstrated to elute early in the gradient (F16-F21, highlighted in the black box) before the activated BoNT molecule (F21-F29). Thus, this shows good separation of the Lys-C from the rBoNT/A1. The max intensity of the fractions appears to be similar to 30 ng/mL Lys-C, suggesting that any residual Lys-C present in the rBoNT/A1 fractions would be less than this concentration.

Example 7—Activation and Final Purification of Recombinant Endopeptidase-Negative Botulinum Neurotoxin Sub-Serotype A2 (rBoNT/A2(0))

(52) Single chain rBoNT/A2(0) was purified by fast protein liquid chromatography (FPLC) using high-performance butyl sepharose hydrophobic interaction chromatography (Butyl HP HIC) for capture followed by an intermediate purification step with high-performance quaternary ammonium sepharose anionic exchange chromatography (Q HP AEC). This molecule was then incubated with 4 μg/mL Lys-C at 37° C. for 2 h to yield the active di-chain.

(53) The Lys-C was resolved from the activated rBoNT/A2(0) using high-performance phenyl sepharose hydrophobic interaction chromatography (PhHP HIC). This involved adjusting the reaction mixture with a high-salt Tris buffer before loading onto the Phenyl HP column, followed by a high-salt wash and subsequent protein elution with a linear gradient to a low-salt Tris buffer. Elution fractions were analysed by denaturing gel electrophoresis (SDS PAGE) and a colorimetric Lys-C activity assay (FIG. 12)—this showed that the Lys-C eluted early in the gradient (F5-F11) just before the activated BoNT molecule (F12-F15). Thus, this column shows good separation of the Lys-C from the rBoNT/A2(0).

Example 8—Activation and Final Purification of Recombinant Endopeptidase-Negative Botulinum Neurotoxin Sub-Serotype A5 (rBoNT/A5(0))

(54) Single chain rBoNT/A5(0) was purified by fast protein liquid chromatography (FPLC) using high-performance butyl sepharose hydrophobic interaction chromatography (Butyl HP HIC) for capture followed by an intermediate purification step with high-performance quaternary ammonium sepharose anionic exchange chromatography (Q HP AEC). This molecule was then incubated with 2.5 μg/mL Lys-C at 37° C. for 2 h to yield the active di-chain. The Lys-C was resolved from the activated rBoNT/A5(0) using high-performance phenyl sepharose hydrophobic interaction chromatography (Phenyl HP HIC). This involved adjusting the reaction mixture with a high-salt Tris buffer before loading onto the Phenyl HP column, followed by a high-salt wash and subsequent protein elution with a linear gradient to a low-salt Tris buffer. Elution fractions were analysed by denaturing gel electrophoresis (SDS PAGE) and a colorimetric Lys-C activity assay (FIG. 13)—this showed that the Lys-C eluted early in the gradient (F10-F39) before the activated BoNT molecule (F51-F65). Thus, this column shows good separation of the Lys-C from the rBoNT/A5(0).

Example 9—Activation and Final Purification of Recombinant Endopeptidase-Negative Botulinum Neurotoxin Sub-Serotype A6 (rBoNT/A6(0))

(55) Single chain rBoNT/A6(0) was purified first by sodium sulphate precipitation and resolubilisation into sodium acetate before capture with fast protein liquid chromatography (FPLC) using high-performance sulphopropyl sepharose cationic exchange chromatography (SP HP CEC) followed by buffer exchange into Tris buffer at pH 8. This molecule was then incubated with 0.3 μg/mL Lys-C at 37° C. for 2 h to yield the active di-chain. The Lys-C was resolved from the activated rBoNT/A6(0) using high-performance phenyl sepharose hydrophobic interaction chromatography (Phenyl HP HIC). This involved adjusting the reaction mixture with a high-salt Tris buffer before loading onto the Phenyl HP column, followed by a high-salt wash and subsequent protein elution with a linear gradient to a low-salt Tris buffer. Elution fractions were analysed by denaturing gel electrophoresis (SDS PAGE) and a colorimetric Lys-C activity assay (FIG. 14)—this showed that the Lys-C eluted early in the gradient (F6-F13) before the activated BoNT molecule (F16-F27). This shows excellent separation of the Lys-C from the rBoNT/A6(0).

Example 10—Formulation Comprising Active Di-Chain BoNT/A Substantially Free from Lys-C

(56) The following six liquid compositions comprising active di-chain BoNT/A are prepared (Table 6).

(57) TABLE-US-00006 TABLE 6 Exemplary BoNT/A formulations 1 2 3 4 5 6 Polysorbate 80 0.10 mg/mL 0.10 mg/mL 0.10 mg/mL 0.10 mg/mL — — Poloxamer — — — — 0.04 mg/mL 0.04 mg/mL Sucrose  4.0 mg/mL —  4.0 mg/mL —  4.0 mg/mL — Mannitol —  4.0 mg/mL —  4.0 mg/mL —  4.0 mg/mL Sodium Chloride 8.76 mg/mL 8.76 mg/mL 8.76 mg/mL 8.76 mg/mL 8.76 mg/mL 8.76 mg/mL pH 6.5 6.5 6.5 6.5 6.5 6.5 Buffer L-Histidine/ L-Histidine/ Di sodium phosphate/ Di sodium phosphate/ L-Histidine/ L-Histidine/ Hydrochloric acid Hydrochloric acid Citric acid anhydrous Citric acid anhydrous Hydrochloric acid Hydrochloric acid Di-Chain BoNT/A  20 ng/mL  20 ng/mL  20 ng/mL  20 ng/mL  20 ng/mL  20 ng/mL MilliQ water q.s. to 1 mL q.s. to 1 mL q.s. to 1 mL q.s. to 1 mL q.s. to 1 mL q.s. to 1 mL

(58) All six compositions are stored at 25° C. for 12 weeks. The stability of the di-chain BoNT/A protease function is assessed during that period using a cell-free endopeptidase assay.

(59) TABLE-US-00007 SEQ ID NO: 1 Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly  1               5                   10                  15  Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro              20                  25                  30  Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg          35                  40                  45  Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu      50                  55                  60  Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr  65                  70                  75                  80  Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu                  85                  90                  95  Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val              100                 105                 110  Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys          115                 120                 125  Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr      130                 135                 140  Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile  145                 150                 155                 160  Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr                  165                 170                 175  Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe              180                 185                 190  Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu          195                 200                 205  Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu      210                 215                 220  Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn  225                 230                 235                 240  Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu                  245                 250                 255  Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys              260                 265                 270  Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn          275                 280                 285  Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val      290                 295                 300  Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys  305                 310                 315                 320  Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu                  325                 330                 335  Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp              340                 345                 350  Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn          355                 360                 365  Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr      370                 375                 380  Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn  385                 390                 395                 400  Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu                  405                 410                 415  Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg              420                 425                 430 Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys         435                 440                 445 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe     450                 455                 460 Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu  465                 470                 475                 480  Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu                  485                 490                 495  Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro              500                 505                 510  Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu          515                 520                 525  Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu      530                 535                 540  Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu  545                 550                 555                 560  His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu                  565                 570                 575  Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys              580                 585                 590  Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu          595                 600                 605  Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr      610                 615                 620  Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala  625                 630                 635                 640  Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu                  645                 650                 655  Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala              660                 665                 670  Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys          675                 680                 685  Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu      690                 695                 700  Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys  705                 710                 715                 720  Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu                  725                 730                 735  Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn              740                 745                 750  Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp          755                 760                 765  Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile      770                 775                 780  Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met  785                 790                 795                 800  Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys                  805                 810                 815  Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly              820                 825                 830  Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp          835                 840                 845  Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser      850                 855                 860  Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn  865                 870                 875                 880  Leu Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser                  885                 890                 895  Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn              900                 905                 910  Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu          915                 920                 925  Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser      930                 935                 940  Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn  945                 950                 955                 960  Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val                  965                 970                 975  Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu              980                 985                 990  Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser          995                 1000                1005  Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg      1010                1015                1020  Leu Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln      1025                1030                1035  Lys Pro Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile      1040                1045                1050  Met Phe Lys Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp      1055                1060                1065  Ile Lys Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu      1070                1075                1080  Ile Lys Asp Leu Tyr Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys      1085                1090                1095  Asp Phe Trp Gly Asp Tyr Leu Gln Tyr Asp Lys Pro Tyr Tyr Met      1100                1105                1110  Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn Asn Val      1115                1120                1125  Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg Gly Ser Val      1130                1135                1140  Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr Arg Gly Thr      1145                1150                1155  Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn Lys Asp Asn Ile      1160                1165                1170  Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val Val Val Lys Asn      1175                1180                1185  Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly Val Glu      1190                1195                1200  Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser      1205                1210                1215  Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn      1220                1225                1230  Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly      1235                1240                1245  Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala      1250                1255                1260  Ser Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu      1265                1270                1275  Gly Cys Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu      1280                1285                1290  Arg Pro Leu      1295  SEQ ID NO: 2 Cys Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly  1               5                   10                  15  Tyr Asn Lys Ala Leu Asn Asp Leu Cys              20                  25  SEQ ID NO: 3 Cys Val Arg Gly Ile Ile Pro Phe Lys Thr Lys Ser Leu Asp Glu Gly  1               5                   10                  15  Tyr Asn Lys Ala Leu Asn Asp Leu Cys              20                  25  SEQ ID NO: 4 Cys Val Arg Gly Ile Ile Pro Phe Lys Thr Lys Ser Leu Asp Glu Gly  1               5                   10                  15  Tyr Asn Lys Ala Leu Asn Asp Leu Cys              20                  25  SEQ ID NO: 5 Cys Val Arg Gly Ile Ile Pro Phe Lys Thr Lys Ser Leu Asp Glu Gly  1               5                   10                  15  Tyr Asn Lys Ala Leu Asn Tyr Leu Cys              20                  25  SEQ ID NO: 6 Cys Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Glu Gly 1               5                   10                  15  Tyr Asn Lys Ala Leu Asn Glu Leu Cys              20                  25  SEQ ID NO: 7 Cys Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Glu Gly  1               5                   10                  15  Tyr Asn Lys Ala Leu Asn Asp Leu Cys              20                  25  SEQ ID NO: 8 Val Gln Gly Gln Ser Val Lys Gly Val Gly Lys Thr Ser Leu Asp Gly  1               5                   10                  15  Leu Val Asn Ile Asp Val Thr Tyr Gly Asn Gly Lys Tyr Tyr Leu Lys              20                  25                  30  Asp Ser Asn Lys Asn Ile Tyr Leu Tyr Asp Leu Lys Asn Gln Val Asp          35                  40                  45  Glu Tyr Asp Leu Tyr Asn Tyr Leu Ser Arg Pro Asn Tyr Lys Gln Ile      50                  55                  60  Leu Met Ser Lys Ser Glu Leu Ile Ser Asn Tyr Asn Asn Asn Phe Ile  65                  70                  75                  80  Ala Asn Asn Gln Val Asn Ser Val Asp Ala Tyr Val Asn Thr Asn Lys                  85                  90                  95  Thr Tyr Asp Tyr Tyr Lys Asn Lys Leu Asn Arg Asn Ser Ile Asp Asn              100                 105                 110  Lys Gly Met Asn Ile Asn Gly Phe Val His Val Gly Arg Asn Tyr Gly          115                 120                 125  Asn Ala Phe Trp Tyr Gly Pro Tyr Asp Gly Met Phe Phe Gly Asp Gly      130                 135                 140  Asp Gly Ile Tyr Phe Ser Ser Leu Ala Lys Ser Leu Asp Val Val Gly  145                 150                 155                 160  His Glu Leu Ser His Gly Val Thr Asn Lys Glu Ser Asn Leu Lys Tyr                  165                 170                 175  Glu Asn Glu Ser Gly Ala Leu Asn Glu Ser Phe Ser Asp Ile Met Gly              180                 185                 190  Val Ala Val Glu Gly Lys Asn Phe Val Leu Gly Glu Asp Cys Trp Val          195                 200                 205  Ala Gly Gly Val Met Arg Asp Met Glu Asn Pro Ser Arg Gly Gly Gln      210                 215                 220  Pro Ala His Met Lys Asp Tyr Lys Tyr Lys Thr Met Asn Asp Asp Asn  225                 230                 235                 240  Gly Gly Val His Thr Asn Ser Gly Ile Ile Asn His Ala Ala Tyr Leu                  245                 250                 255  Val Ala Asp Gly Ile Glu Lys Thr Gly Ala Lys Asn Ser Lys Asp Ile              260                 265                 270  Met Gly Lys Ile Phe Tyr Thr Ala Asn Cys Tyr Lys Trp Asp Glu Thr          275                 280                 285  Thr Asn Phe Ala Lys Cys Arg Asn Asp Val Val Gln Val Thr Lys Glu      290                 295                 300  Leu Tyr Gly Glu Asn Ser Asn Tyr Val Lys Ile Val Glu Lys Ala Phe  305                 310                 315                 320  Asp Gln Val Gly Ile Thr Ala Thr Pro Gln Leu Pro Leu                  325                 330  SEQ ID NO: 9 Met Lys Ser Lys Lys Leu Leu Ala Thr Val Leu Ser Ala Val Ile Thr  1               5                   10                  15  Phe Ser Thr Val Ser Ala Val Tyr Ala Ala Pro Val Gly Lys Glu Ser              20                  25                  30  Lys Val Glu Pro Lys Thr Thr Thr Ile Thr Trp Glu Lys Asn Glu Gln          35                  40                  45  Asn Thr Lys Lys Ala Ala Thr Asp Ile Thr Glu Lys Lys Phe Asn Asn      50                  55                  60  Ser Glu Glu Ile Thr Lys Phe Phe Glu Lys Asn Ile Ser Lys Phe Gly  65                  70                  75                  80  Val Gln Lys Gly Ser Leu Lys Asn Thr Lys Thr Val Lys Asp Glu Lys                  85                  90                  95  Gly Lys Thr Asn Tyr His Met Ile Tyr Glu Val Glu Gly Ile Pro Val              100                 105                 110  Tyr Tyr Gly Arg Ile Val Phe Thr Thr Glu Lys Asp Ser Ser Met Asp          115                 120                 125  Ser Ile Asn Gly Arg Ile Asp Thr Val Phe Glu Asn Gly Asn Trp Lys      130                 135                 140  Asn Lys Ile Lys Leu Ser Lys Glu Asp Ala Ile Ala Lys Ala Lys Asn  145                 150                 155                 160  Asp Ile Lys Asp Glu Lys Ala Thr Ser Lys Lys Thr Asp Leu Tyr Leu                  165                 170                 175  Tyr Asn Phe Glu Gly Lys Pro Tyr Val Val Tyr Leu Val Asp Leu Ile              180                 185                 190  Thr Asp Asn Gly Ser Trp Thr Val Phe Val Asn Ala Glu Asp Gly Ser          195                 200                 205  Ile Val Asn Lys Phe Asn Asn Thr Pro Thr Leu Ile Asp Thr Lys Asp      210                 215                 220  Gln Lys Leu Pro Asn Ala Lys Lys Ile Lys Asp Glu Ala Lys Lys Ala  225                 230                 235                 240  Ser Asn Ala Asn Asn Val Ile Asp Val Gln Gly Gln Ser Val Lys Gly                  245                 250                 255  Val Gly Lys Thr Ser Leu Asp Gly Leu Val Asn Ile Asp Val Thr Tyr              260                 265                 270  Gly Asn Gly Lys Tyr Tyr Leu Lys Asp Ser Asn Lys Asn Ile Tyr Leu          275                 280                 285  Tyr Asp Leu Lys Asn Gln Val Asp Glu Tyr Asp Leu Tyr Asn Tyr Leu      290                 295                 300  Ser Arg Pro Asn Tyr Lys Gln Ile Leu Met Ser Lys Ser Glu Leu Ile  305                 310                 315                 320  Ser Asn Tyr Asn Asn Asn Phe Ile Ala Asn Asn Gln Val Asn Ser Val                  325                 330                 335  Asp Ala Tyr Val Asn Thr Asn Lys Thr Tyr Asp Tyr Tyr Lys Asn Lys              340                 345                 350  Leu Asn Arg Asn Ser Ile Asp Asn Lys Gly Met Asn Ile Asn Gly Phe          355                 360                 365  Val His Val Gly Arg Asn Tyr Gly Asn Ala Phe Trp Tyr Gly Pro Tyr      370                 375                 380  Asp Gly Met Phe Phe Gly Asp Gly Asp Gly Ile Tyr Phe Ser Ser Leu  385                 390                 395                 400  Ala Lys Ser Leu Asp Val Val Gly His Glu Leu Ser His Gly Val Thr                  405                 410                 415  Asn Lys Glu Ser Asn Leu Lys Tyr Glu Asn Glu Ser Gly Ala Leu Asn              420                 425                 430  Glu Ser Phe Ser Asp Ile Met Gly Val Ala Val Glu Gly Lys Asn Phe          435                 440                 445  Val Leu Gly Glu Asp Cys Trp Val Ala Gly Gly Val Met Arg Asp Met      450                 455                 460  Glu Asn Pro Ser Arg Gly Gly Gln Pro Ala His Met Lys Asp Tyr Lys  465                 470                 475                 480  Tyr Lys Thr Met Asn Asp Asp Asn Gly Gly Val His Thr Asn Ser Gly                  485                 490                 495  Ile Ile Asn His Ala Ala Tyr Leu Val Ala Asp Gly Ile Glu Lys Thr              500                 505                 510  Gly Ala Lys Asn Ser Lys Asp Ile Met Gly Lys Ile Phe Tyr Thr Ala          515                 520                 525  Asn Cys Tyr Lys Trp Asp Glu Thr Thr Asn Phe Ala Lys Cys Arg Asn      530                 535                 540  Asp Val Val Gln Val Thr Lys Glu Leu Tyr Gly Glu Asn Ser Asn Tyr  545                 550                 555                 560  Val Lys Ile Val Glu Lys Ala Phe Asp Gln Val Gly Ile Thr Ala Thr                  565                 570                 575  Pro Gln Leu Pro Leu              580  SEQ ID NO: 10 atggttcaag gtcaaagcgt taaaggagta ggaaaaacta gcttggatgg actagtaaat   60  attgatgtaa cttatggaaa tggaaaatac tatttaaaag atagcaacaa aaatatttat  120  ctatatgact taaaaaatca agttgatgaa tatgatctat acaattatct tagtagacct  180  aactataaac aaatattaat gagcaaatct gaattaatat ctaattacaa taataatttt  240  atagccaaca atcaggttaa ttctgtagat gcttatgtaa acacaaataa aacctatgat  300  tattataaaa acaaattaaa tagaaacagt attgataata agggtatgaa tattaatggg  360  tttgttcatg taggtagaaa ttatggtaat gctttttggt acggtccata tgatgggatg  420  ttctttggcg atggcgacgg aatatacttc tcttcccttg caaaatcttt agatgttgta  480  ggccacgaat taagtcatgg tgtaacaaat aaagagtcta atcttaaata tgaaaatgaa  540  tctggtgccc taaatgaatc tttctcagat attatgggag tagctgttga gggtaaaaac  600  tttgtactag gtgaagattg ctgggttgct ggaggagtaa tgagagatat ggaaaatcca  660  tccagaggag gccaaccagc tcatatgaaa gattataaat acaaaactat gaatgacgat  720  aacggtggtg ttcatacaaa ttcaggtata ataaaccatg ctgcttattt agttgcagat  780  ggaatagaaa aaactggtgc aaaaaatagt aaagatatta tgggaaaaat attctataca  840  gctaattgct ataaatggga tgaaacaaca aattttgcta agtgcagaaa tgatgtagtc  900  caagttacta aagaacttta tggcgaaaat agcaactatg taaaaattgt tgaaaaagct  960  tttgaccaag ttggaataac tgctacacct caattaccat tataa                 1005  SEQ ID NO: 11 atgaaaagta aaaaattatt agctacagtg ctaagtgccg tgatcacttt ttctactgtt   60  tctgcagttt atgctgcgcc tgtaggaaaa gaaagtaaag ttgaaccaaa aactacaaca  120  ataacttggg aaaaaaatga acaaaatact aaaaaagctg ctactgatat aactgaaaag  180  aaatttaaca attctgagga gataactaaa ttctttgaaa aaaatatatc taaatttggt  240  gtacaaaaag gttctcttaa aaacaccaag actgtaaaag acgaaaaagg taaaactaac  300  tatcatatga tttatgaagt agaaggtata cctgtatact atggaagaat tgtttttaca  360  actgaaaaag actcctccat ggattctata aacggtagaa ttgatactgt ttttgaaaat  420  gggaattgga aaaacaaaat caaactatca aaagaagatg ctatagcaaa agctaaaaat  480  gatattaaag atgaaaaagc aactagtaaa aagaccgatt tatatctgta taattttgag  540  ggcaaacctt atgtagttta tttagtagat ctaattacag acaacgggag ttggacggtt  600  ttcgttaatg ctgaggatgg ttctatagta aataaattta ataatactcc tactttaatt  660  gatactaaag atcaaaaatt acccaatgct aaaaaaatta aagatgaagc taaaaaagct  720  agtaatgcaa ataatgtaat tgatgttcaa ggtcaaagcg ttaaaggagt aggaaaaact  780  agcttggatg gactagtaaa tattgatgta acttatggaa atggaaaata ctatttaaaa  840  gatagcaaca aaaatattta tctatatgac ttaaaaaatc aagttgatga atatgatcta  900  tacaattatc ttagtagacc taactataaa caaatattaa tgagcaaatc tgaattaata  960  tctaattaca ataataattt tatagccaac aatcaggtta attctgtaga tgcttatgta 1020  aacacaaata aaacctatga ttattataaa aacaaattaa atagaaacag tattgataat 1080  aagggtatga atattaatgg gtttgttcat gtaggtagaa attatggtaa tgctttttgg 1140  tacggtccat atgatgggat gttctttggc gatggcgacg gaatatactt ctcttccctt 1200  gcaaaatctt tagatgttgt aggccacgaa ttaagtcatg gtgtaacaaa taaagagtct 1260  aatcttaaat atgaaaatga atctggtgcc ctaaatgaat ctttctcaga tattatggga 1320  gtagctgttg agggtaaaaa ctttgtacta ggtgaagatt gctgggttgc tggaggagta 1380  atgagagata tggaaaatcc atccagagga ggccaaccag ctcatatgaa agattataaa 1440  tacaaaacta tgaatgacga taacggtggt gttcatacaa attcaggtat aataaaccat 1500  gctgcttatt tagttgcaga tggaatagaa aaaactggtg caaaaaatag taaagatatt 1560  atgggaaaaa tattctatac agctaattgc tataaatggg atgaaacaac aaattttgct 1620  aagtgcagaa atgatgtagt ccaagttact aaagaacttt atggcgaaaa tagcaactat 1680  gtaaaaattg ttgaaaaagc ttttgaccaa gttggaataa ctgctacacc tcaattacca 1740  ttataa                                                            1746  SEQ ID NO: 12 Val Pro Pro Thr Pro Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys  1               5                   10                  15