BoNT / a2 mutants and uses thereof

Abstract

The application belongs to the technical field of bioengineering and relates to screening and application of a botulinum neurotoxin type A2 (BoNT / A2) mutant. Compared with wild-type BoNT / A2, the mutant comprises at least one of the following amino acid mutation sites: S954N, K1137F, S1142M, V1143Y, V1144R, T1153M, T1153Q, T1153R, E1156K or E1156M. The mutant has the advantages of good biological activity, high safety window and fast onset. The application also relates to a production process of a recombinant botulinum toxin, and the process is simple and easy to obtain high-purity recombinant botulinum toxin.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, and in particular to a BoNT / A2 mutant and its applications. Background Technology

[0002] Clostridium botulinum, also known as botulinum toxin, is a bacterium widely distributed in nature that produces botulinum toxin (BoNT) under anaerobic conditions. Its molecular weight is 150 kDa, composed of a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC), linked by a disulfide bond. BoNT inhibits the release of acetylcholine (ACh) in the peripheral nervous system, thereby paralyzing muscles.

[0003] Seven different serotypes of botulinum toxin (BoNT / AG) have been identified, and these serotypes differ in the severity and duration of infection and paralysis. Considering factors such as paralysis rate, safety, and duration (Toxins 2018, 10, 268), botulinum toxin type A (BoNT / A) is the preferred choice for clinical application. However, botulinum toxin type A also has several subtypes, BoNT / A1-A6. Which subtype has the greatest development potential?

[0004] Christina L. Pier et al. (FEBS Letters 585 (2011) 199–206) reported that BoNT / A2 can be internalized into nerve cells at a faster rate, and the LD50 of BoNT / A1 and BoNT / A2 are 1.3 x 10⁻⁶. 8 Units / mg, 4.3 x 10 8 The unit count / mg indicates that BoNT / A2 is more potent than A1. Yasushi Torii et al. (Toxicon 57 (2011) 93–99) also demonstrated in mice that BoNT / A2 has higher potency, longer duration of action, and better safety than BoNT / A1. Norio Akaike et al. (J Physiol 591.4 (2013)) showed that BoNT / A2 has a faster and stronger muscle paralysis effect and better safety than BoNT / A1. Multiple clinical trials in healthy volunteers and patients with muscle spasms have also demonstrated that BoNT / A2 has better clinical efficacy and safety than BoNT / A1 and onabotulinumtoxin A (BOTOX) (Y. Mukai et al. / Toxicon 81 (2014) 32–36). These data all indicate that BoNT / A2 has greater clinical development value than BoNT / A1.

[0005] In addition, considering that botulinum toxin is the most potent neurotoxin known to date, its safety has always been a top concern. High toxicity and low diffusivity are two important research directions. (1) The higher the toxicity, the lower the dosage and the lower the potential immunogenicity. Toxicity mainly depends on the enzymatic cleavage ability of the botulinum toxin light chain, and there is little research on this aspect. (2) The lower the diffusivity of botulinum toxin, the lower the impact on other organs. Diffusivity mainly depends on the affinity between the botulinum toxin heavy chain and the receptor protein / ganglioside on the membrane. Studies have shown (Int.J.Mol.Sci.2023,24(6),5690) that the potency of BoNT / A4 is 1000 times lower than that of BoNT / A1. However, there is no significant difference in the enzymatic activity of the two subtypes of light chains. If the amino acids in the receptor-binding domain of BoNT / A4 are mutated to the same amino acids as those in BoNT / A1, the potency of BoNT / A4 can reach a level comparable to that of BoNT / A1. Furthermore, Min Dong et al. (NatCommun. 2017 Jul 3; 8(1):53) used bacterial adenylate cyclase based two-hybrid (BACTH) to screen a library of mutants of the BoNT / B heavy chain receptor binding domain, obtaining the E1191M / S1199Y mutant combination with high affinity for the receptor hSytII. Compared with the wild type, this mutant combination showed an 11-fold increase in the inhibitory effect on neurotransmission. Subsequently, they (PLoS Biol 18(3):e3000618) also found that the I1248W / V1249W mutant combination related to BoNT / B upper membrane lipid binding could also improve the therapeutic window. Patent CN119708179A also obtained some BoNT / A1 mutants using a similar BACTH technology. This evidence suggests that enhancing the affinity between botulinum toxin and its specific receptor can effectively reduce drug diffusion and improve the therapeutic window. However, these library screening methods are time-consuming and the experimental process is complex. Artificial intelligence methods have shown great application potential in the prediction, analysis, and design of biomolecular structures. Cell-free protein expression technology is a novel in vitro protein expression technique with significant advantages such as high throughput and automation. Artificial intelligence and cell-free protein expression technology form a complete "design-validation" closed loop, which can greatly accelerate the development of innovative botulinum toxins. This invention utilizes this development logic to complete the upgrade and iteration of BoNT / A2 in a short period of time, obtaining mutants with better biological activity.

[0006] Existing methods for preparing botulinum toxin mainly fall into two categories: one involves anaerobic fermentation extraction of botulinum toxin using Clostridium botulinum; the other involves expression of recombinant botulinum toxin using bacteria or other microorganisms. The first method can produce botulinum toxin-containing complex proteins of different molecular weights, such as 900kDa, 450kDa, and 150kDa, by controlling the production process. However, this method is complex, lengthy, and the final product contains impurities and requires extremely strict production conditions. Therefore, expressing recombinant botulinum toxin using bacteria has become the mainstream research trend.

[0007] Recombinant expression can be further divided into single-peptide chain recombinant expression and double-peptide chain recombinant expression. Naturally occurring botulinum toxin consists of two polypeptide chains (heavy and light chains) linked by a disulfide bond. Therefore, single-peptide chain expression requires adding activation sites between the light and heavy chains, and then artificially activating them during subsequent purification. Patent CN119823215A uses primers at the C-terminus of the light chain and the N-terminus of the heavy chain for two restriction enzyme digestion sites. This single-peptide chain double digestion method leaves extra amino acids at the C-terminus of the light chain, resulting in a sequence difference from natural botulinum toxin. Double-peptide chain expression can be further subdivided into inclusion body renaturation and enzyme digestion activation methods. Patent CN114989271A uses an inclusion body renaturation production method. After expressing the heavy and light chains separately, purification requires seven steps: denaturation, renaturation, hydrophobic chromatography, ammonium sulfate precipitation, dialysis, DEAE anion exchange chromatography, and G25M molecular sieve chromatography, taking five days and being a lengthy process. Patent CN115785237A uses a single-enzyme digestion method for the dipeptide chain, adding a TEV cleavage site before the heavy chain. This construction method requires the first amino acid at the N-terminus of the heavy chain to be glycine, resulting in the loss of amino acid residues before glycine in the final product, causing partial amino acid deletion. Patent CN116813727A uses a single-peptide chain double-enzyme digestion method, which requires sequential activation with SUMO-tagged enzyme and thrombin. This activation method leaves residual cleavage sites on the final protein product. Scientific research (Biochimie (1990) 72, 661-664) shows that natural BoNT / A undergoes a post-translational modification process, requiring sequential cleavage activation at two sites: E444-G445 and K448-A449, ultimately forming the mature form of BoNT / A with the N-terminal starting amino acid 449A. This mature form of BoNT / A has higher biological activity. Therefore, the sequences obtained by patents such as CN119823215A, CN115785237A, and CN116813727A are inconsistent with natural botulinum toxin, or contain missing amino acids or residual enzyme cleavage sites, all of which may affect the biological activity of botulinum toxin. The process method of this application can perfectly solve the above problems. Summary of the Invention

[0008] Numerous studies have demonstrated that BoNT / A2 has a faster onset of action and higher safety profile than the currently marketed BoNT / A1. Increasing the affinity of BoNT / A2 for the receptor protein SV2C is an effective method to enhance its biological activity. Therefore, in this application, based on wild-type BoNT / A2, several BoNT / A2 mutants with high affinity for the receptor protein SV2C were rapidly screened and obtained using artificial intelligence prediction and cell-free high-throughput protein expression technology. The mutation sites described in this invention are also applicable to other botulinum toxins that use SV2C as a receptor.

[0009] This application provides a BoNT / A2 mutant, relative to wild-type BoNT / A2, wherein the BoNT / A2 mutant includes at least one of the following amino acid mutation sites: S954N, K1137F, S1142M, V1143Y, V1144R, T1153M, T1153Q, T1153R, E1156K, or E1156M, and the amino acid sequence of the wild-type BoNT / A2 is shown in SEQ ID NO:1.

[0010] This application also provides an isolated polynucleotide encoding the aforementioned BoNT / A2 mutant.

[0011] This application also provides an expression vector containing the polynucleotides isolated above.

[0012] This application also provides an expression system containing the above-described expression vector or genome in which exogenous polynucleotides described above are integrated.

[0013] This application also provides a pharmaceutical composition comprising the above-described BoNT / A2 mutant and a pharmaceutically acceptable carrier or excipient.

[0014] This application also provides the use of the above-mentioned BoNT / A2 mutant, polynucleotide, expression vector, expression system or pharmaceutical composition in the preparation of pharmaceuticals, cosmetic products or targeted drug delivery vectors.

[0015] This application also provides a method for expressing recombinant botulinum toxin or a mutant.

[0016] (1) The heavy and light chains of the recombinant botulinum toxin mutant are expressed as two separate polypeptide chains in the same engineered bacterium, and then the assembly of the complete botulinum toxin is automatically completed; and

[0017] (2) The amino acid sequence of the first polypeptide chain, from the N-terminus to the C-terminus, consists of a protein tag sequence (SEQ ID NO:2), a recombinant botulinum toxin, or the heavy chain of a mutant; and

[0018] (3) The amino acid sequence of the second polypeptide chain is the light chain of recombinant botulinum toxin or a mutant.

[0019] Recombinant expression of botulinum toxin has become a mainstream research trend, but currently available methods all have some shortcomings. This application provides a method for preparing botulinum toxin, in which the heavy and light chains of botulinum toxin are expressed separately as two polypeptide chains in the same engineered bacteria, completing the assembly of complete botulinum toxin. The dual-peptide expression method used in this application can be achieved by constructing the heavy and light chains separately onto their respective plasmid vectors and then co-transforming them into bacteria; or by constructing the heavy and light chains onto different multiple cloning sites (MSCs) of the same plasmid vector. The latter is simpler to operate and is a better choice, which is described in this invention as an example. For example, the first chain is the botulinum toxin heavy chain, with a 6x His-sumo tag fused to its N-terminus. The senp2 enzyme recognizes the three-dimensional structure of the sumo protein and cleaves it at a specific site. The advantages of this production method are: (1) No excess amino acids remain at the N-terminus of the heavy chain, and it can ensure that A449 is the starting amino acid, which is completely consistent with the mature natural botulinum toxin; (2) senp2 has extremely high recognition specificity and will not produce non-specific cleavage; (3) The purification process is simple, and only 1-2 simple nickel affinity chromatography steps are needed to obtain high-purity botulinum toxin.

[0020] In summary, the recombinant expression protocol used in this application completely mimics the post-translational modification process of natural botulinum toxin, ultimately yielding a protein product with an amino acid sequence completely identical to that of natural botulinum toxin. This expression protocol has a high upper limit, applicable to both wild-type botulinum toxin and the preparation of botulinum toxin mutants. Attached Figure Description

[0021] This application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein:

[0022] Figure 1 Schematic diagram of wild-type BoNT / A2 heavy chain mutant expression in a cell-free protein synthesis system as detected by SDS-PAGE;

[0023] Figure 2 Schematic diagram of each component during the purification process of SV2C-LD4 by SDS-PAGE detection;

[0024] Figure 3Schematic diagram of the components during the purification process of BoNT / A2(874-1296)-K1137F, V1143Y, V1144R, T1153M, T1153Q and E1156K mutants by SDS-PAGE detection;

[0025] Figure 4 Schematic diagram of the construction of three common clones expressing botulinum toxin in this invention;

[0026] Figure 5 Schematic diagram of each component during the purification process of BoNT / A2 by SDS-PAGE detection;

[0027] Figure 6 Schematic diagram of the components during the purification process of BoNT / A2-E1156K, detected by SDS-PAGE and gel filtration chromatography;

[0028] Figure 7 Mass spectrometry determination of the N-terminal amino acid sequences of the BoNT / A2 and BoNT / A2-E1156K heavy chains;

[0029] Figure 8 Mass spectrometry determination of the C-terminal amino acid sequences of BoNT / A2 and BoNT / A2-E1156K heavy chains;

[0030] Figure 9 Mass spectrometry determination of the N-terminal amino acid sequences of the BoNT / A2 and BoNT / A2-E1156K light chains;

[0031] Figure 10 Mass spectrometry determination of the C-terminal amino acid sequences of the BoNT / A2 and BoNT / A2-E1156K light chains;

[0032] Figure 11 Schematic diagram of the average body weight of mice in each dose group after injection of full-length BoNT / A2 and BoNT / A2-E1156K;

[0033] Figure 12 : ED50 curves of half-effective dose for each dose group after injection of full-length BoNT / A2 and BoNT / A2-E1156K;

[0034] Figure 13 : Toe abduction score (DAS) curves of mice in each dose group within the first 72 hours after injection of full-length BoNT / A2 and BoNT / A2-E1156K. Detailed Implementation

[0035] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0036] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0037] Flowcharts are used in this specification to illustrate the operations performed by the system according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to these processes, or one or more steps can be removed from them.

[0038] This application provides a BoNT / A2 mutant, relative to wild-type BoNT / A2, wherein the BoNT / A2 mutant includes at least one of the following amino acid mutation sites: S954N, K1137F, S1142M, V1143Y, V1144R, T1153M, T1153Q, T1153R, E1156K, or E1156M, and the amino acid sequence of the wild-type BoNT / A2 is shown in SEQ ID NO:1.

[0039] The term "sequence" in this article should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding the amino acid sequence, unless a more specific interpretation is required in this article.

[0040] In some embodiments, the BoNT / A2 mutant may include at least one of the following amino acid mutation sites relative to wild-type BoNT / A2: V1143Y, V1144R, T1153M, T1153Q, or E1156K.

[0041] This application also provides an isolated polynucleotide encoding the aforementioned BoNT / A2 mutant.

[0042] As used herein, the terms “polynucleotide,” “nucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to nucleic acids including DNA, RNA, their derivatives, or combinations thereof.

[0043] This application also provides an expression vector containing the isolated polynucleotides described above.

[0044] As used herein, "expression vector" refers to a polynucleotide capable of carrying at least one polynucleotide fragment. An expression vector can deliver fragments of nucleic acids, or individual polynucleotides, into a host cell. It may contain at least one expression cassette containing a regulatory sequence for the proper expression of the polynucleotide incorporated therein. The polynucleotide to be introduced into the cell (e.g., a polynucleotide encoding a target product or a selectable marker) can be inserted into the expression cassette of the vector for expression therefrom. Expression vectors according to this application may be in circular or linear (linearized) form and also include expression vector fragments. The term "expression vector" also includes artificial chromosomes or similar individual polynucleotides that allow the transfer of exogenous nucleic acid fragments.

[0045] This application also provides an expression system containing the above-described expression vector or a genome in which exogenous polynucleotides described above are integrated.

[0046] In some embodiments, the host cell of the expression system may be selected from eukaryotic or prokaryotic cells. In some embodiments, preferably, the host cell may be selected from any one of *Escherichia coli*, *Klebsiella pneumoniae*, *Bacteroides ovalis*, *Campylobacter jejuni*, *Staphylococcus saprophyticus*, *Enterococcus faecalis*, *Bacteroides pleomorphus*, *Bacteroides var. var. var.*, *Bacteroides moniliforme*, *Lactobacillus casei*, *Bacteroides fragilis*, *Acinetobacter rumenii*, *Fusobacterium nucleatum*, *Bacteroides johnsonii*, *Bacteroides thaliana*, *Lactobacillus rhamnosus*, *Bacteroides masei*, *Bacteroides foetida*, *Fusobacterium death*, or *Bifidobacterium breve*. In some embodiments, more preferably, the expression system may be selected from *Escherichia coli*. In some embodiments, further preferably, the expression system may be selected from any one of *E. coli* BL21 Star (DE3), *origami*, *Rosetta* (DE3), *Origami B* (DE3), BL21 CodonPlus (DE3), or SHuffle T7.

[0047] This application also provides a pharmaceutical composition comprising the above-described BoNT / A2 mutant and a pharmaceutically acceptable carrier or excipient.

[0048] The term “pharmaceutically acceptable” as used in this article generally means a compound, material, composition, and / or dosage form that is suitable, within reasonable medical judgment, for contact with human and animal tissues, organs, and / or body fluids without excessive toxicity, irritation, allergic response, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0049] The excipients include various excipients and diluents, which are not essential active ingredients and do not cause excessive toxicity after application. The excipients contain sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), or binders. The excipients also contain other low molecular weight peptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol, or sorbitol. When used in an aqueous solution for injection, the excipients are selected from physiological saline, isotonic glucose solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannitol or sugar alcohol isotonic solution. The aqueous solution for injection contains a solubilizer. The solubilizer is selected from alcohols (ethanol), polyols (propylene glycol or PEG), and / or nonionic surfactants (Tween 80 or HCO-50).

[0050] In the pharmaceutical composition provided in this application, the BoNT / A2 mutant can be a single active ingredient, or it can be combined with one or more other active ingredients that are useful for the treatment of diseases to form a combined formulation.

[0051] The content of the active ingredient in the pharmaceutical composition is a safe and effective amount, which should be adjustable by those skilled in the art. For example, the dosage of the BoNT / A2 mutant and the active ingredient in the pharmaceutical composition depends on the patient's weight, the type of application, the condition and severity of the disease.

[0052] This application also provides the use of the above-mentioned BoNT / A2 mutant, polynucleotide, expression vector, expression system or pharmaceutical composition in the preparation of pharmaceuticals, cosmetic products or targeted drug delivery vectors.

[0053] In some embodiments, the drug may be a medication for treating one or more of the following conditions: neuromuscular diseases, chronic pain, glandular secretion disorders, or urinary system dysfunction. In some embodiments, the neuromuscular disease may include muscle spasms or dystonia.

[0054] This application also provides a method for expressing recombinant botulinum toxin or a mutant, (1) expressing the heavy and light chains of the recombinant botulinum toxin mutant as two polypeptide chains in the same engineered bacteria, and then automatically assembling the complete botulinum toxin; and

[0055] (2) The amino acid sequence of the first polypeptide chain, from the N-terminus to the C-terminus, consists of a protein tag sequence (SEQ ID NO:2), a recombinant botulinum toxin, or the heavy chain of a mutant; and

[0056] (3) The amino acid sequence of the second polypeptide chain is the light chain of recombinant botulinum toxin or a mutant.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0058] Example 1: Design of Botulinum toxin type A2 (BoNT / A2) mutant

[0059] The complex structure PDB:6ESI of the BoNT / A2 heavy chain and SV2C luminal domain 4 (LD4) was input into the PISA interface (https: / / www.ebi.ac.uk / msd-srv / prot_int / cgi-bin / piserver) to analyze the amino acids on BoNT / A2 involved in the interaction with SV2C. Thirteen amino acids on BoNT / A2 were found to be involved in SV2C binding. Then, the complex structure PDB:6ESI was input into the mutant prediction AI program, and the mutated site was selected. The system automatically evaluated the changes in parameters such as ΔDDG and ΔDDE after mutating this site to 19 other amino acids. Approximately 300 mutants were evaluated for parameter changes. From mutants with ΔDDG less than 0, combined with manual screening, 28 mutants were selected for further wet experimental validation (see Table 1).

[0060] Table 1. List of high-affinity mutants predicted by artificial intelligence methods

[0061] Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8 S954N K955A K1137F S1142E V1143Y V1144M T1153K E1156K K955I K1137N S1142I V1144R T1153M E1156M K955L S1142L T1153Q E1156R K955M S1142M T1153R K955Q S1142Q K955S S1142R K955Y S1142T S1142V

[0062] Example 2: Construction of BoNT / A2 heavy chain mutant using the Gibson assembly method

[0063] Using Q45894 from Uniport as the wild-type BoNT / A2 sequence (SEQ ID NO:1), the gene segment 874-1296 (SEQ ID NO:4) was synthesized and cloned into the psumo vector to express Sumo3-BoNT / A2 HC (874-1296). Forward and reverse primers for the mutants were designed (Table 2), and amplification was performed using KOD neo-plus (Toyobo, Japan) (Table 3). After digestion with 1 μL of DpnI (NEB) enzyme at 68°C for 1 hour in each reaction, ligation was performed using the Novizan ClonExpress kit (Table 4) to obtain different mutants.

[0064] Table 2 Primer sequences for mutant construction

[0065]

[0066]

[0067] Table 3 PCR Procedure

[0068]

[0069] Table 4 Connection Program

[0070] Connecting components Volume 5x CEII buffer 4uL Dpn1 digestion products 10uL PCR grade water 4uL Exnase II 2uL

[0071] Example 3: Preparation of recombinant BoNT / A2 heavy chain mutant using cell-free protein expression technology

[0072] Using the ligation product from Example 2 as a template, linear DNA (Fwd: GCGAAATTAATACGACTCACTATAGGGGAATTGTG (SEQ ID NO: 77); Rev: CAAAAAACCCCTCAAGACCC GTTTAGAGG (SEQ ID NO: 78)) was obtained by PCR amplification. An appropriate amount of MST09 bacterial extract was thawed to room temperature, and 50 mM iodoacetamide was added and incubated at room temperature for 30 minutes. Then, 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids (1 mM tyrosine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine, 15 mM potassium phosphate, and 200 nM MT7 were added. RNAP and 20 nM linear DNA were reacted at 180 rpm and 30 °C for 8 hours. The supernatant was then collected by centrifugation, and SDS-PAGE was used to detect the expression of each mutant. Figure 1). The experimental method is referenced from Efficacy of a Potential Trivalent Vaccine Basedon Hc Fragments of Botulinum Toxins A,B,and E Produced in a Cell-FreeExpression System, CLINICAL AND VACCINE IMMUNOLOGY, May 2010, P784–792.

[0073] Results analysis: The theoretical molecular weight of BoNT / A2 fused with the tag protein and its heavy chain mutant is 60.7 kDa. After Coomassie blue staining, clear expression bands can be seen at the theoretical molecular weight positions on the gel.

[0074] Example 4: Expression and purification of recombinant SV2C

[0075] Cloning: Based on the uniport ID: Q496J9 human SV2C sequence (SEQ ID NO:15), the gene segment 474-567 corresponding to luminaldomain4 (LD4) was synthesized and cloned into the psumo vector to express Sumo3-SV2C-LD4 (SEQ ID NO:16). The plasmid was then transformed into BL21(DE3) competent bacteria.

[0076] Culture: Select a single colony of Sumo-SV2C-LD4 and inoculate it into 100 ml of 2YT medium containing ampicillin resistance. Incubate overnight at 37°C and 220 rpm. Inoculate the overnight bacterial culture at a 1:100 volume ratio and expand the culture at 37°C and 200 rpm. After approximately 4 hours, when the absorbance at 600 nm (OD600) is between 0.6 and 0.8, cool the culture to 20°C and add 0.5 mM isopropyl β-D-thiogalactoside (IPTG), then continue incubation for 16 hours.

[0077] Purification: Bacterial cells were collected by centrifugation at 4500 rpm for 20 min. The cells were resuspended in Buffer A (20 mM Tris-HCl, pH 7.4, 0.3 M NaCl, 20 mM imidazole) and then lysed using a low-temperature high-pressure homogenizer. After lysis, the sample was centrifuged at 40000 g for 60 min, and the supernatant was collected. The sample was then loaded onto a nickel column (Hitrap, Cytiva) at a flow rate of 5 ml / min, washed with Buffer A until the UV280 baseline stabilized, and eluted with Buffer B (20 mM Tris-HCl, pH 7.4, 0.3 M NaCl, 200 mM imidazole). Protein purity and homogeneity were assessed using SDS-PAGE. Figure 2 Left).

[0078] Enzyme digestion: Senp2 enzyme was added to the elution buffer at a ratio of 1:100 and dialyzed against 20 mM Tris-HCl, pH 7.4, 0.3 M NaCl. The next day, the solution was passed through a nickel column again, and the flow-through was collected. The solution was washed with dialysate, rinsed with buffer A, and eluted with buffer B. The protein purity and homogeneity of each fraction were determined using SDS-PAGE. Figure 2 right).

[0079] Results analysis: After two steps of nickel bead affinity chromatography, SV2C-LD4 protein with high purity was obtained.

[0080] Example 5: Preliminary screening of recombinant BoNT / A2 heavy chain mutants

[0081] Dilute SV2C-LD4 protein to 10 μg / ml with carbonate (pH 9.6) buffer, add 100 μL of protein to each well of the ELISA plate, and incubate overnight at 4°C. Discard the liquid from the wells and wash three times with PBST buffer (containing 0.05% Tween-20d PBS). Next, add 200 μL of 5% skim milk to each well as a blocking buffer and incubate at room temperature for 1–2 hours. After washing three times with PBST, add 100 μL of analytical sample (cell-free protein reaction supernatant serially diluted 1:1, 1:5, 1:10, 1:20, 1:40, 1:80, 1:160, 1:320) to each well. Incubate at room temperature for 2 hours and wash four times with PBST. Dilute anti-HIS-HRP (ProteinTech) 1:10000 with 5% BSA and add 100 μL to each well. After incubating at room temperature for 1 hour, wash 3-5 times with PBST to ensure complete removal of unbound antibodies. Finally, add 100 μL of TMB substrate (Beyotime, P0208) to each well and incubate at room temperature in the dark for 20 minutes. Read the absorbance values ​​at 370 nm using a microplate reader (MD SpectraMax iD3) (Table 5).

[0082] Results analysis: This experiment was conducted in two 96-well plates. After comparing the OD370 absorbance values ​​with those of the wild type, the mutants K1137F, V1143Y, V1144R, T1153M, T1153Q, and E1156K were selected for purification and affinity characterization experiments.

[0083] Table 5. OD370 readings for preliminary screening of BoNT / A2 heavy chain mutants and SV2C-LD4 by ELISA.

[0084]

[0085] Example 6: Purification of recombinant BoNT / A2 heavy chain mutant

[0086] For detailed purification procedures of the BoNT / A2(874-1296)-K1137F, V1143Y, V1144R, T1153M, T1153Q, and E1156K mutants, please refer to Example 4. The purity and homogeneity of each protein component were detected using SDS-PAGE. Figure 3 ).

[0087] Results analysis: After two-step nickel bead affinity chromatography, high-purity BoNT / A2(874-1296)-K1137F, V1143Y, V1144R, T1153M, T1153Q and E1156K mutant proteins were obtained.

[0088] Example 7: Determination of affinity between BoNT / A2 mutant and SV2C-LD4

[0089] The instrument used in this experiment was an Octet R8, and the chip used was an NTA sensor. 200 μL of Kbuffer (containing 0.2% BSA and 0.002% Tween 20 PBS) was added to each well of a 96-well plate. The probe was oscillated at 1000 rpm during the experiment. The BoNT / A2 mutant was diluted to five gradients: 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM. sumo3-SV2C-LD4 was diluted to 5 μg / mL. The experiment was conducted according to the systems listed in Table 6 below:

[0090] Table 6

[0091] Step Assay time(s) Buffer 1. Baseline 60 K buffer 2. Loading 20 sumo3-SV2C-LD4 3. Baseline2 60 K buffer 4. Association 120 BoNT / A2 5. Dissociation 120 K buffer

[0092] Data analysis and fitting were performed using Octet analysis, with a 1:1 fitting mode. Affinity test results are shown in Table 7.

[0093] Results analysis: Except for K1137F, the affinity of the other mutants and SV2C-LD4 was higher than that of the wild type. The next step is to prepare the full-length BoNT / A2 and BoNT / A2-E1156K proteins.

[0094] Table 7. Affinity test results between BoNT / A2 heavy chain mutant and sumo3-SV2C-LD4

[0095] KD(nM) ka(1 / Ms) kdis(1 / s) KD(WT) / KD(mutant) WT 145.6 1.849E05 2.693E-02 1 K1137F 301.3 2.190E05 6.598E-02 0.48 V1143Y 124.1 2.639E05 3.276E-02 1.168 V1144R 88.44 3.479E05 2.033E-02 1.65 T1153M 62.4 3.168E05 1.977E-02 2.32 T1153Q 119.7 1.177E05 1.409E-02 1.22 E1156K 28.2 2.921E05 8.261E-03 5.16

[0096] Example 8: Preparation of full-length BoNT / A2

[0097] The present invention uses a single-enzyme digestion method for dipeptide chains, which allows for different cloning and construction methods. Figure 4 The following examples use 4B as an example. Using Q45894 from Uniport as the wild-type BoNT / A2 sequence (SEQ ID NO:1), amino acid sequences 449-1296 were synthesized and cloned into multiple cloning site 1 (MCS1) of the psumo vector, and amino acid sequences 1-444 were synthesized and cloned into multiple cloning site 2 (MCS2) of the psumo vector to express the full-length BoNT / A2 (SEQ ID NO:17), whose corresponding DNA sequence is (SEQ ID NO:18). Two-step nickel bead affinity chromatography was used to purify the protein; detailed experimental procedures are described in Example 4. SDS-PAGE was used to detect the purity and homogeneity of each protein component. Figure 5 ).

[0098] Results analysis: High-purity BoNT / A2 protein was obtained through a simple two-step nickel bead affinity chromatography.

[0099] Example 9: Preparation of full-length BoNT / A2-E1156K

[0100] Based on the gene sequence used in Example 7, and referring to the mutant construction method in Example 2, BoNT / A2-E1156K (SEQ ID NO: 19) was obtained using primers (Fwd: CCCTCTATaAAGGTACCAAATTCATTATTAAAAAATACG; Rev: GGTACCTTtATAGAGGGTCGAGTTCAGGTAAATG), and its corresponding DNA sequence is (SEQ ID NO: 20). Two-step nickel bead affinity chromatography was used to purify the protein; detailed experimental procedures are described in Example 4. Finally, gel filtration chromatography (Supeedex 200) was used to identify protein purity, with PBS as the buffer. SDS-PAGE was used to detect the purity and homogeneity of each protein component. Figure 6 ).

[0101] Results analysis: High-purity BoNT / A2-E1156K was obtained after two-step nickel bead affinity chromatography. Gel filtration chromatography results also showed that the two-step nickel bead affinity chromatography yielded high-purity and homogeneous BoNT / A2-E1156K.

[0102] Example 10: Identification of the N-terminal and C amino acid sequences of the full-length BoNT / A2 and BoNT / A2-E1156K heavy chains by mass spectrometry

[0103] The purified BoNT / A2 and BoNT / A2-E1156K samples were separated into heavy and light chains by SDS-PAGE gel electrophoresis. The heavy chain strips were then subjected to disulfide bond reduction with dithiothreitol (DTT) (10 mM, 37℃, 1 h) followed by alkylation with iodoacetamide (IAA) (20 mM, room temperature, protected from light, 30 min) to stabilize the free thiol groups. Protein samples were digested using two or more proteases (e.g., Trypsin, Lys-C) (enzyme:substrate ratio 1:20–1:50, 37℃, 12–16 h) to generate a mixture of peptides covering the N-terminal region. The digested peptides were then desalted and purified using a C18 column. Raw data were generated using ultra-high performance liquid chromatography (UHPLC) with a C18 reversed-phase column and a high-resolution tandem mass spectrometer (Orbitrap Fusion Lumos). The raw mass spectrometry data were processed using software such as MaxQuant and ProteomeDiscoverer, and data analysis was performed by comparing the BoNT / A2 sequence (SEQ ID NO:1). Experimental data were exported using PEAKS.

[0104] Results analysis: The sequences of the BoNT / A2 and BoNT / A2-E1156K heavy chain N-terminal peptide fragments read by mass spectrometry are:

[0105] ALNDLCIK (SEQ ID NO:79) has four different modification types, possibly due to introduction during mass spectrometry experiments (Table 6). Figure 7 This demonstrates that the enzyme digestion method used in this invention is highly efficient and specific, and the N-terminal peptide sequence is consistent with theoretical predictions and completely identical to the N-terminal sequence of the naturally occurring mature form, BoNT / A. The mass spectrometry readings of BoNT / A2 and...

[0106] The C-terminal peptide sequence of the BoNT / A2-E1156K heavy chain is RTFGCSWEFIPVDDGWGESSL (SEQ ID NO:80), which is completely identical to the C-terminal sequence of the naturally occurring mature form of BoNT / A2 (Table 8). Figure 8 ).

[0107] Table 8. Mass spectrometry analysis results of N-terminal and C-terminal peptides of the BoNT / A2 and BoNT / A2-E1156K heavy chains.

[0108]

[0109] Example 11: Identification of the N-terminal and C amino acid sequences of the full-length BoNT / A2 and BoNT / A2-E1156K light chains using mass spectrometry

[0110] For the specific experimental procedure, please refer to Example 10.

[0111] Results analysis: The N-terminal peptide sequences of the BoNT / A2 and BoNT / A2-E1156K light chains read by mass spectrometry are PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVK ((SEQ ID NO:81)) (Table 7, Figure 9 The C-terminal peptide sequences of the BoNT / A2 and BoNT / A2-E1156K light chain read by mass spectrometry are GIIPFKTKSLDE (SEQ ID NO:82), which is consistent with theoretical predictions and completely consistent with the C-terminal sequence of the naturally occurring mature form of BoNT / A2 (Table 9). Figure 10 ).

[0112] Table 9. Mass spectrometry analysis results of N-terminal and C-terminal peptides of the BoNT / A2 and BoNT / A2-E1156K light chains.

[0113] Peptide -10LgP Mass ppm m / z RT PFVNKQFNYKDPVNGVDIAYIK(SEQ ID NO:83) 118.66 2568.3376 0.0 857.1198 39.34 PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVK 118.45 3731.9497 0.9 747.3979 45.74 GIIPFKTKSLDE 117.88 1346.7445 0.1 674.3795 30.11

[0114] Example 12: Lethality study of full-length BoNT / A2 and BoNT / A2-E1156K

[0115] This study used clean-grade female Kunming mice weighing 18-22g. The injected drugs were full-length BoNT / A2 and...

[0116] BoNT / A2-E1156K. First, all drugs were diluted with PBS containing 0.25% BSA to prepare concentrations of 1000 pg, 500 pg, 250 pg, 125 pg, 100 pg, 62.5 pg, 50 pg, 25 pg, 12.5 pg, and 6.25 pg of toxin per 10 μL. Mice were randomly divided into 10 groups of 4 mice each. Each mouse received a 10 μL intramuscular injection (IM) into the gastrocnemius muscle of its left hind limb. Survival was recorded for 4 days to determine the intramuscular injection median lethal dose (IMLD50).

[0117] Results analysis: All patients in the 50 pg dose group of the full-length BoNT / A2 died, while all patients in the 25 pg dose group survived, indicating that the IMLD50 is between 25 pg and 50 pg. All patients in the 25 pg dose group of the full-length BoNT / A2-E1156K ...

[0118] Example 13: Bioactivity study of full-length BoNT / A2 and BoNT / A2-E1156K

[0119] This study used clean-grade female Kunming mice weighing 18-22g. The injected drugs were full-length BoNT / A2 and...

[0120] BoNT / A2-E1156K. First, all drugs were diluted with PBS containing 0.25% BSA to prepare concentrations of 9 pg, 3 pg, 1 pg, 0.33 pg, and 0.11 pg toxin per 100 μL. Mice were then randomly divided into 5 groups of 4 mice each. Each mouse was weighed and marked before injection, and 10 μL of the drug was injected intramuscularly into the gastrocnemius muscle of the left hind limb. The first DAS score was performed 6 hours after injection, and scores were then recorded every 24 hours thereafter, with mouse weight recorded daily. The highest dose group (9 pg / mouse) in this experiment corresponds to 0.18 U–0.36 U of BoNT / A2 and 0.36 U–0.72 U of BoNT / A2-E1156K.

[0121] The DAS (Desquamation and Abduction Score) is a physiological model for assessing muscle relaxation. Its basic principle is that when mice are briefly suspended by their tails, they exhibit a typical startle response, which in turn induces hind limb extension and toe abduction. The model uses a 5-point scale (0-4 points). The DAS scoring criteria are as follows: 0 points: flat foot, toes are the same as the untreated leg; 1 point: flat foot, abduction width differs from the untreated leg or two toes touch, the rest is completely spread out; 2 points: slight gaps between the tips of all toes, or three toes touch; 3 points: if the foot is flat, five toes touch; if the foot is bent, four toes touch; 4 points: bent foot, five toes touch.

[0122] Results analysis: 1. The average body weight of mice in each dose group of BoNT / A2 and BoNT / A2-E1156K steadily increased after 14 days, indicating that the mice tolerated these two drugs well. Figure 11 2. Using the highest average DAS score for each dose as the y-axis and the corresponding toxin dosage logarithmically transformed to base 3 as the x-axis, a nonlinear regression curve was plotted. The dose corresponding to a DAS of 2 is the ED50. The results show that the ED50 of BoNT / A2 is 2 pg, and the ED50 of BoNT / A2-E1156K is 0.33 pg. Figure 12This means that BoNT / A2-E1156K requires only 1 / 6 the dose of BoNT / A2 to achieve the same muscle paralysis effect as BoNT / A2, indicating that the high affinity of BoNT / A2-E1156K for the receptor SV2C translates into better biological activity in vivo. 3. The DAS score curves of each dose group of BoNT / A2 and BoNT / A2-E1156K showed a clear dose-dependent relationship, and the scores of each dose group of BoNT / A2-E1156K were significantly higher than those of BoNT / A2, further confirming that BoNT / A2-E1156K has a better inhibitory effect on nerve impulse transmission. 4. Both BoNT / A2 and BoNT / A2-E1156K dose groups began to take effect 6 hours after injection (the earliest assessment time point), reaching peak effect after 24 hours. Figure 13 The results from Toxicon 107(2015)37e42, Toxins2020,12,393, Clinical, Cosmetic and Investigational Dermatology 2023:16 581–591 and Toxins2025,17,230 all show that BoNT / A1 takes effect as early as 10 hours after injection and reaches its peak at 48-60 hours. This further demonstrates the advantages of BoNT / A2 and BoNT / A2-E1156K compared to BoNT / A1, with faster onset and shorter time to peak effect.

[0123] SEQ ID NO:1BoNT / A2 WT

[0124] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVP

[0125] VSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQP

[0126] DGSYRSEELNLVIIGPSADIIQFECKSFGHDVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGA

[0127] GKFATDPAVTLAHELIHAEHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQEN

[0128] EFRLYYYNKFKDVASTLNKAKSIIGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTE

[0129] DNFVNFFKVINRKTYLNFDKAVFRINIPDENYTIKDGFNLKGANLSTNFNGQNTEINSRNFTRLKNFTGL

[0130] FEFYKLLCVRGIIPFKTKSLDEGYNKALNDLCIKVNNWDLFFSPSEDNFTTNDLDKVEEITADTNIEAAEENI

[0131] SLDLIQQYYLTDFDFDNEPENISIENLSSDIIGQLEPMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGDSR

[0132] IILTNSAEEALLKPNVAYTFFSSKYVKKINKAVEAFMFNLNWAEELVYDFTDETNEVTTMDKAIDITIIVPYIG

[0133] PALNIGNMLSKGEFVEAIIFTGVVAMLEFIPEYALPVFGTFAIVSYIANKVLTVQTINNALSKRNEKWDEVY

[0134] KYTVTNWLAKVNTQIDLIREKMKKALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINSAM

[0135] ININKFLDQCSVSYLMNSMIPYAVKRLKDFDASVRDVLLKYIYDNRGTLVLQVDRLKDEVNNTLSADIPF

[0136] QLSKYVDNKKLLSTFTTEYIKNIVNTSILSIVYKKDDLIDLSRYGAKINIGDRVYYDSIDKNQIKLINLESSTIEV

[0137] ILKNAIVYNSMYENFSTSFWIKIPKYFSKINLNNEYTIINCIENNSGWKVSLNYGEIIWTLQDNKQNIQRVV

[0138] FKYSQMVNISDYINRWIFVTITNNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDPRRYIMIKY

[0139] FNLFDKELNEKEIKDLYDSQSNSGILKDFWGNYLQYDKPYYMLNLFDPNKYVDVNNIGIRGYMYLKGPR

[0140] GSVVTTNIYLNSTLYEGTKFIIKKYASGNEDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIP

[0141] DVGNLSQVVVMKSKDDQGIRNKCKMNLQDNNGNDIGFIGFHLYDNIAKLVASNWYNRQVGKASRTFGCSWEFIPVDDGWGESSL*

[0142] SEQ ID NO:2fusion protein

[0143] MGHHHHHHGSLQEEKPKEGVKTENDHINLKVAGQDGSVVQFKIKRHTPLSKLMKAYCERQGLSMRQIRFRFDGQPINETDTPAQLEMEDEDTIDVFQQQTGG*

[0144] SEQ ID NO:3BoNT / A2 LC

[0145] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVP

[0146] VSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQP

[0147] DGSYRSEELNLVIIGPSADIIQFECKSFGHDVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGA

[0148] GKFATDPAVTLAHELIHAEHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQEN

[0149] EFRLYYYNKFKDVASTLNKAKSIIGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTE

[0150] DNFVNFFKVINRKTYLNFDKAVFRINIVPDENYTIKDGFNLKGANLSTNFNGQNTEINSRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDE*

[0151] SEQ ID NO:4BoNT / A2 HC(449-1296)WT

[0152] VNTSILSIVYKKDDLIDLSRYGAKINIGDRVYYDSIDKNQIKLINLESSTIEVILKNAIVYNSMYENFSTSFWI

[0153] KIPKYFSKINLNNEYTIINCIENNSGWKVSLNYGEIIWTLQDNKQNIQRVVFKYSQMVNISDYINRWIFVTIT

[0154] NNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDPRRYIMIKYFNLFDKELNEKEIKDLYDSQS

[0155] NSGILKDFWGNYLQYDKPYYMLNLFDPNKYVDVNNIGIRGYMYLKGPRGSVVTTNIYLNSTLYEGTKFII

[0156] KKYASGNEDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKDDQGIRNKCKMNLQDNNGNDIGFIGFHLYDNIAKLVASNWYNRQVGKASRTFGCSWEFIPVDDGWGESSL*SEQ ID NO:5 BoNT / A2 HC(449-1296)S954N

[0157] VNTSILSIVYKKDDLIDLSRYGAKINIGDRVYYDSIDKNQIKLINLESSTIEVILKNAIVYNSMYENFSTSFWI

[0158] KIPKYFNKINLNNEYTIINCIENNSGWKVSLNYGEIIWTLQDNKQNIQRVVFKYSQMVNISDYINRWIFVTI

[0159] TNNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDPRRYIMIKYFNLFDKELNEKEIKDLYDSQ

[0160] SNSGILKDFWGNYLQYDKPYYMLNLFDPNKYVDVNNIGIRGYMYLKGPRGSVVTTNIYLNSTLYEGTKFI

[0161] IKKYASGNEDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKDDQGIRNKCKMNLQDNNGNDIGFIGFHLYDNIAKLVASNWYNRQVGKASRTFGCSWEFIPVDDGWGESSL*SEQ IDNO:6 BoNT / A2 HC(449-1296)K1137F

[0162] VNTSILSIVYKKDDLIDLSRYGAKINIGDRVYYDSIDKNQIKLINLESSTIEVILKNAIVYNSMYENFSTSFWI

[0163] KIPKYFSKINLNNEYTIINCIENNSGWKVSLNYGEIIWTLQDNKQNIQRVVFKYSQMVNISDYINRWIFVTIT

[0164] NNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDPRRYIMIKYFNLFDKELNEKEIKDLYDSQS

[0165] NSGILKDFWGNYLQYDKPYYMLNLFDPNKYVDVNNIGIRGYMYLFGPRGSVVTTNIYLNSTLYEGTKFII

[0166] KKYASGNEDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKDDQGIRNKCKMNLQDNNGNDIGFIGFHLYDNIAKLVASNWYNRQVGKASRTFGCSWEFIPVDDGWGESSL*SEQ ID NO:7 BoNT / A2 HC(449-1296)S1142M

[0167] VNTSILSIVYKKDDLIDLSRYGAKINIGDRVYYDSIDKNQIKLINLESSTIEVILKNAIVYNSMYENFSTSFWI

[0168] KIPKYFSKINLNNEYTIINCIENNSGWKVSLNYGEIIWTLQDNKQNIQRVVFKYSQMVNISDYINRWIFVTIT

[0169] NNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDPRRYIMIKYFNLFDKELNEKEIKDLYDSQS

[0170] NSGILKDFWGNYLQYDKPYYMLNLFDPNKYVDVNNIGIRGYMYLKGPRGMVVTTNIYLNSTLYEGTKFII

[0171] KKYASGNEDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKDDQGIRNKCKMNLQDNNGNDIGFIGFHLYDNIAKLVASNWYNRQVGKASRTFGCSWEFIPVDDGWGESSL*SEQ ID NO:8 BoNT / A2 HC(449-1296)V1143Y

[0172] VNTSILSIVYKKDDLIDLSRYGAKINIGDRVYYDSIDKNQIKLINLESSTIEVILKNAIVYNSMYENFSTSFWI

[0173] KIPKYFSKINLNNEYTIINCIENNSGWKVSLNYGEIIWTLQDNKQNIQRVVFKYSQMVNISDYINRWIFVTIT

[0174] NNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDPRRYIMIKYFNLFDKELNEKEIKDLYDSQS

[0175] NSGILKDFWGNYLQYDKPYYMLNLFDPNKYVDVNNIGIRGYMYLKGPRGSYVTTNIYLNSTLYEGTKFII

[0176] KKYASGNEDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKDDQGIRNKCKMNLQDNNGNDIGFIGFHLYDNIAKLVASNWYNRQVGKASRTFGCSWEFIPVDDGWGESSL*SEQ ID NO:9 BoNT / A2 HC(449-1296)V1144R

[0177] VNTSILSIVYKKDDLIDLSRYGAKINIGDRVYYDSIDKNQIKLINLESSTIEVILKNAIVYNSMYENFSTSFWI

[0178] KIPKYFSKINLNNEYTIINCIENNSGWKVSLNYGEIIWTLQDNKQNIQRVVFKYSQMVNISDYINRWIFVTIT

[0179] NNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDPRRYIMIKYFNLFDKELNEKEIKDLYDSQS

[0180] NSGILKDFWGNYLQYDKPYYMLNLFDPNKYVDVNNIGIRGYMYLKGPRGSVRTTNIYLNSTLYEGTKFII

[0181] KKYASGNEDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKDDQGIRNKCKMNLQDNNGNDIGFIGFHLYDNIAKLVASNWYNRQVGKASRTFGCSWEFIPVDDGWGESSL*SEQ ID NO:10 BoNT / A2 HC(449-1296)T1153M

[0182] VNTSILSIVYKKDDLIDLSRYGAKINIGDRVYYDSIDKNQIKLINLESSTIEVILKNAIVYNSMYENFSTSFWI

[0183] KIPKYFSKINLNNEYTIINCIENNSGWKVSLNYGEIIWTLQDNKQNIQRVVFKYSQMVNISDYINRWIFVTIT

[0184] NNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDPRRYIMIKYFNLFDKELNEKEIKDLYDSQS

[0185] NSGILKDFWGNYLQYDKPYYMLNLFDPNKYVDVNNIGIRGYMYLKGPRGSVVTTNIYLNSMLYEGTKFII

[0186] KKYASGNEDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKDDQGIRNKCKMNLQDNNGNDIGFIGFHLYDNIAKLVASNWYNRQVGKASRTFGCSWEFIPVDDGWGESSL*SEQ ID NO:11 BoNT / A2 HC(449-1296)T1153Q

[0187] VNTSILSIVYKKDDLIDLSRYGAKINIGDRVYYDSIDKNQIKLINLESSTIEVILKNAIVYNSMYENFSTSFWI

[0188] KIPKYFSKINLNNEYTIINCIENNSGWKVSLNYGEIIWTLQDNKQNIQRVVFKYSQMVNISDYINRWIFVTIT

[0189] NNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDPRRYIMIKYFNLFDKELNEKEIKDLYDSQS

[0190] NSGILKDFWGNYLQYDKPYYMLNLFDPNKYVDVNNIGIRGYMYLKGPRGSVVTTNIYLNSQLYEGTKFII

[0191] KKYASGNEDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKDDQGIRNKCKMNLQDNNGNDIGFIGFHLYDNIAKLVASNWYNRQVGKASRTFGCSWEFIPVDDGWGESSL*SEQ ID NO:12 BoNT / A2 HC(449-1296)T1153R

[0192] VNTSILSIVYKKDDLIDLSRYGAKINIGDRVYYDSIDKNQIKLINLESSTIEVILKNAIVYNSMYENFSTSFWI

[0193] KIPKYFSKINLNNEYTIINCIENNSGWKVSLNYGEIIWTLQDNKQNIQRVVFKYSQMVNISDYINRWIFVTIT

[0194] NNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDPRRYIMIKYFNLFDKELNEKEIKDLYDSQS

[0195] NSGILKDFWGNYLQYDKPYYMLNLFDPNKYVDVNNIGIRGYMYLKGPRGSVVTTNIYLNSRLYEGTKFII

[0196] KKYASGNEDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKDDQGIRNKCKMNLQDNNGNDIGFIGFHLYDNIAKLVASNWYNRQVGKASRTFGCSWEFIPVDDGWGESSL*SEQ ID NO:13 BoNT / A2 HC(449-1296)E1156K

[0197] VNTSILSIVYKKDDLIDLSRYGAKINIGDRVYYDSIDKNQIKLINLESSTIEVILKNAIVYNSMYENFSTSFWI

[0198] KIPKYFSKINLNNEYTIINCIENNSGWKVSLNYGEIIWTLQDNKQNIQRVVFKYSQMVNISDYINRWIFVTIT

[0199] NNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDPRRYIMIKYFNLFDKELNEKEIKDLYDSQS

[0200] NSGILKDFWGNYLQYDKPYYMLNLFDPNKYVDVNNIGIRGYMYLKGPRGSVVTTNIYLNSTLYKGTKFII

[0201] KKYASGNEDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKDDQGIRNKCKMNLQDNNGNDIGFIGFHLYDNIAKLVASNWYNRQVGKASRTFGCSWEFIPVDDGWGESSL*SEQ ID NO:14 BoNT / A2 HC(449-1296)E1156M

[0202] VNTSILSIVYKKDDLIDLSRYGAKINIGDRVYYDSIDKNQIKLINLESSTIEVILKNAIVYNSMYENFSTSFWI

[0203] KIPKYFSKINLNNEYTIINCIENNSGWKVSLNYGEIIWTLQDNKQNIQRVVFKYSQMVNISDYINRWIFVTIT

[0204] NNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDPRRYIMIKYFNLFDKELNEKEIKDLYDSQS

[0205] NSGILKDFWGNYLQYDKPYYMLNLFDPNKYVDVNNIGIRGYMYLKGPRGSVVTTNIYLNSTLYMGTKFII

[0206] SV2C

[0207] MEDSYKDRTSLMKGAKDIAREVKKQTVKKVNQAVDRAQDEYTQRSYSRFQDEEDDDDYYPAGETYN

[0208] GEANDDEGSSEATEGHDEDDEIYEGEYQGIPSMNQAKDSIVSVGQPKGDEYKDRRELESERRADEEE

[0209] LAQQYELIIQECGHGRFQWALFFVLGMALMADGVEVFVVGFVLPSAETDLCIPNSGSGWLGSIVYLGM

[0210] MVGAFFWGGLADKVGRKQSLLICMSVNGFFAFLSSFVQGYGFFLFCRLLSGFGIGGAIPTVFSYFAEVL

[0211] AREKRGEHLSWLCMFWMIGGIYASAMAWAIIPHYGWSFSMGSAYQFHSWRVFVIVCALPCVSSVVAL

[0212] TFMPESPRFLLEVGKHDEAWMILKLIHDTNMRARGQPEKVFTVNKIKTPKQIDELIEIESDTGTWYRRCF

[0213] VRIRTELYGIWLTFMRCFNYPVRDNTIKLTIVWFTLSFGYYGLSVWFPDVIKPLQSDEYALLTRNVERDK

[0214] YANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDF

[0215] EPYKFIDSEFKNCSFFHNKTGCQITFDDDYSAYWIYFVNFLGTLAVLPGNIVSALLMDRIGRLTMLGGSM

[0216] VLSGISCFFLWFGTSESMMIGMLCLYNGLTISAWNSLDVVTVELYPTDRRATGFGFLNALCKAAAVLGNLIFGSLVSITKSIPILLASTVLVCGGLVGLCLPDTRTQVLM*

[0217] SEQ ID NO:16 sumo-human SV2C-LD4

[0218] MGHHHHHHGSLQEEKPKEGVKTENDHINLKVAGQDGSVVQFKIKRHTPLSKLMKAYCERQGLSMRQI

[0219] RFRFDGQPINETDTPAQLEMEDEDTIDVFQQQTGGERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFKNCSFFHNKT*

[0220] SEQ ID NO:17 Sumo3-HC:LC

[0221] MGHHHHHHGSLQEEKPKEGVKTENDHINLKVAGQDGSVVQFKIKRHTPLSKLMKAYCERQGLSMRQI

[0222] RFRFDGQPINETDTPAQLEMEDEDTIDVFQQQTGGALNDLCIKVNNWDLFFSPSEDNFTNDLDKVEEIT

[0223] ADTNIEAAEENISLDLIQQYYLTFDFDNEPENISIENLSSDIIGQLEPMPNIERFPNGKKYELDKYTMFHYL

[0224] RAQEFEHGDSRIILTNSAEEALLKPNVAYTFFSSKYVKKINKAVEAFMFLNWAEELVYDFTDETNEVTTM

[0225] DKIADITIIVPYIGPALNIGNMLSKGEFVEAIIFTGVVAMLEFIPEYALPVFGTFAIVSYIANKVLTVQTINNAL

[0226] SKRNEKWDEVYKYTVTNWLAKVNTQIDLIREKMKKALENQAEATKAIINYQYNQYTEEEKNNINFNIDDL

[0227] SSKLNESINSAMININKFLDQCSVSYLMNSMIPYAVKRLKDFDASVRDVLLKYIYDNRGTLVLQVDRLKD

[0228] EVNNTLSADIPFQLSKYVDNKKLLSTFTEYIKNIVNTSILSIVYKKDDLIDLSRYGAKINIGDRVYYDSIDKN

[0229] QIKLINLESSTIEVILKNAIVYNSMYENFSTSFWIKIPKYFSKINLNNEYTIINCIENNSGWKVSLNYGEIIWT

[0230] LQDNKQNIQRVVFKYSQMVNISDYINRWIFVTITNNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLD

[0231] GCRDPRRYIMIKYFNLFDKELNEKEIKDLYDSQSNSGILKDFWGNYLQYDKPYYMLNLFDPNKYVDVNN

[0232] IGIRGYMYLKGPRGSVVTTNIYLNSTLYEGTKFIIKKYASGNEDNIVRNNDRVYINVVVKNKEYRLATNAS

[0233] QAGVEKILSALEIPDVGNLSQVVVMKSKDDQGIRNKCKMNLQDNNGNDIGFIGFHLYDNIAKLVASNWYNRQVGKASRTFGCSWEFIPVDDGWGESSL*

[0234] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVP

[0235] VSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHDVLNLTRNGY*

[0236] SEQ ID NO:18 Sumo3-HC:LC DNA sequence

[0237] ATGGGTCATCACCATCATCATCACGGGTCCCTGCAGGAGGAGAAGCCCAAGGAGGGTGTGAAGA

[0238] CAGAGAATGACCACATCAACCTGAAGGTGGCCGGGGCAGGACGGCTCCGTGGTGCAGTTCAAGAT

[0239] CAAGAGGCACACGCCGCTGAGCAAGCTGATGAAGGCCTACTGCGAGAGGCAGGGCTTGTCAATG

[0240] AGGCAGATCAGATTCAGGTTCGACGGGCAGCCAATCAATGAAACTGACACTCCAGCACAGCTGGA

[0241] GATGGAGGACGAGGACACCATCGACGTGTTCCAGCAGCAGACGGGAGGAGCGCTGAATGATTTA

[0242] TGCATTAAAGTTAACAACTGGGATCTGTTTTTAGCCCGAGCGAAGATAACTTTACCAATGACCTGG

[0243] ATAAAGTGGAAGAAATTACCGCGGAATACCAATATTGAAGCGGCGGAAGAAAACATTAGCCTGGATC

[0244] TGATTCAACAGTACTACCTGACCTTTGATTTTGATAATGAACCGGAAACATTAGTATTGAAAACCT

[0245] GAGCAGCGATATTATTGGTCAGCTGGAACCGATGCCGAATATCGAACGCTTTCCGAACGGCAAAA

[0246] AATATGAACTGGATAAATATACGATGTTCCATTACCTGCGCGCGCAGGAATTTGAACATGGCGATT

[0247] CGCGCATTATTCTGACCAACAGCGCGGAAGAGGCGCTGCTGAAACCGAATGTGGCCTACACCTTC

[0248] TTCAGCAGCAAATACGTCAAAAAAATTAATAAAGCGGTGGAAGCGTTTATGTTTCTGAACTGGGCG

[0249] GAAGAGCTGGTATACGATTTTACCGATGAAACCAACGAAGTGACCACTATGGATAAAATTGCAGAC

[0250] ATCACCATTATTGTGCCGTATATTGGCCCGGCTCTGAATATTGGCAATATGCTGAGCAAAGGCGAA

[0251] TTTGTGGAAGCCATTATTTTTACCGGCGTGGTTGCGATGCTGGAATTTATTCCGGAATATGCCCTG

[0252] CCGGTGTTCGGCACCTTTGCGATTGTGAGCTACATCGCCAACAAAGTTCTGACCGTGCAGACCATT

[0253] AACAATGCGCTGAGCAAACGCAATGAAAAATGGGATGAAGTGTACAAATATACGGTAACTAATTGG

[0254] CTGGCGAAAGTTAACACCCAGATCGATCTGATTCGCGAAAAAATGAAAAAAGCGCTGGAAAACCA

[0255] GGCGGAAGCGACCAAGGCGATCATTAACTACCAGTACAACCAGTATACCGAAGAAGAAAAAAACA

[0256] ATATCAATTTTAATATCGATGATCTGAGCAGCAAACTGAACGAATCAATTAACAGCGCGATGATTAA

[0257] CATTAACAAATTTCTGGATCAGTGCAGCGTTAGTTATCTGATGAACAGCATGATTCCGTACGCCGT

[0258] GAAACGCCTGAAAGATTTCGATGCGTCGGTGCGCGATGTGCTGCTGAAATATATCTACGATAATCG

[0259] TGGCACCCTGGTGCTGCAGGTGGATCGCTTAAAAGATGAAGTGAACAATACCCTGAGCGCGGATA

[0260] TTCCGTTTCAGCTGTCGAAATACGTGGATAACAAGAAACTGCTGAGCACCTTTACCGAATATATCAA

[0261] AAATATTGTGAACACCAGCATTCTGAGCATCGTGTACAAAAAAGATGATCTGATTGATCTGAGCCG

[0262] CTATGGTGCCAAAATTAATATTGGTGATCGTGTGTACTACGATTCTATTGATAAAAATCAGATTAAA

[0263] CTGATTAATCTGGAAAGCTCGACCATTGAAGTGATCCTGAAAAATGCGATTGTGTATAATAGCATGT

[0264] ATGAAAACTTCAGCACCAGCTTTTGGATTAAAATTCCGAAATACTTCAGCAAAATTAATCTGAATAA

[0265] CGAATACACAATTATTAATTGCATTGAAAACAATAGCGGCTGGAAAGTGAGCCTGAACTATGGCGA

[0266] GATTATTTGGACCCTGCAGGACAACAAACAGAACATTCAGCGCGTTGTGTTTAAATATAGCCAGAT

[0267] GGTGAATATTAGCGATTATATTAATCGCTGGATCTTTGTGACGATAACCAATAACCGTCTGACCAAA

[0268] AGCAAAATTTATATTAACGGCCGTCTGATTGATCAGAAACCGATTAGCAACCTGGGCAACATTCAT

[0269] GCCAGCAACAAAATTATGTTCAAACTGGATGGCTGCCGCGATCCGCGTCGTTATATTATGATCAAA

[0270] TATTTTAACCTGTTTGATAAAGAACTGAACGAAAAAGAAATCAAAGATTTATATGATAGCCAGAGCA

[0271] ATAGCGGTATTCTGAAAGATTTCTGGGGCAATTATCTGCAGTATGATAAACCGTACTATATGCTGAA

[0272] CCTGTTTGATCCGAACAAATATGTGGATGTGAACAACATTGGCATTCGTGGCTATATGTATCTGAAA

[0273] GGTCCGCGTGGCTCGGTAGTGACCACCAACATTTACCTGAACTCGACCCTCTATGAAGGTACCAA

[0274] ATTCATTATTAAAAAATACGCGAGCGGCAACGAAGATAATATTGTGCGCAACAATGATCGTGTGTAT

[0275] ATTAACGTGGTGGTGAAAAATAAAGAATACCGCCTGGCGACGAATGCAAGCCAGGCGGGCGTGG

[0276] AAAAAATTCTGAGCGCGCTGGAAATTCCGGATGTGGGCAACCTGAGTCAGGTGGTTGTGATGAAA

[0277] TCTAAAGATGATCAGGGTATTCGCAATAAATGTAAAATGAACCTGCAGGATAATAATGGCAATGATA

[0278] TCGGCTTCATTGGTTTTCATCTGTATGACAACATTGCGAAACTGGTGGCGAGCAACTGGTATAATC

[0279] GTCAAGTGGGCAAAGCGAGCCGCACCTTTGGCTGCAGCTGGGAATTTATTCCGGTGGATGATGGC

[0280] TGGGGTGAAAGCAGCCTGTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAG

[0281] CAATAACTAGCATAACCCCTTGGGGCTCTAAACGGGTCTTGAGGGGTTTTTTGCGATCCCGCGAA

[0282] ATTAATACGACTCACTATAGGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTT

[0283] AACTTTAAGAAGGAGATATACCATGCCGTTTGTGAACAAACAGTTTAACTACAAAGATCCTGTTTAAT

[0284] GGCGTTGATATTGCGTACATTAAATTCCGAACGCCGGTCAGATGCAGCCAGTGAAAGCGTTTAAA

[0285] ATTCATAATAAAATTGGGTGATTCCGGAACGCGATACGTTTACCAACCCGGAAGAGGTGATCTG

[0286] AACCCGCCGCGGAAGCGAAGCAGGTGCCGGTGAGCTACTATGATTCGACCTACCTGAGCACGG

[0287] ATAACGAAAAGATAACTATCTGAAAGGCGTGACCAAACTGTTTGAACGCATTTACAGCACCGATCC

[0288] TGGGCCGCATGCTGCTGACCAGCATTGTTCGCGGCATTCCGTTTTGGGGTGGCAGCACGATTGAT

[0289] ACGGAACTGAAAGTGATCGATACCAATTGCATTAATGTGATTCAGCCGGATGGCTCATACCGCAGC

[0290] GAAGAACTGAACCTGGTGATTATTGGCCCGAGCGCGGATATCATTCAGTTTGAATGCAAATCTTTT

[0291] GGCCATGATGTGCTGAATCTGACCCGCAATGGCTACGGCTCGACCCAGTATATTCGCTTTTCACCT

[0292] GATTTTACCTTTGGCTTTGAAGAGAGCCTGGAAGTGGATACCAACCCGCTGCTGGGTGCGGGCAA

[0293] ATTTGCCACCGATCCGGCCGTTACCCTGGCCCATGAACTGATTCATGCCGAACATCGTCTGTATGG

[0294] CATTGCGATTAATCCGAACCGTGTGTTTAAAGTGAATACCAACGCGTATTACGAAATGAGTGGCCT

[0295] GGAAGTGTCGTTTGAAGAACTGCGCACCTTTGGCGGCCATGATGCGAAATTTATTGATAGCCTGCA

[0296] GGAAAATGAATTTCGCCTGTACTATTATAACAAATTTAAAGATGTTGCGAGCACCCTGAATAAAGCG

[0297] AAAAGCATCATTGGCACCACCGCCAGCCTGCAGTACATGAAAAATGTGTTCAAAGAAAAATACCTG

[0298] CTGAGCGAAGATACCAGCGGCAAATTTAGCGTGGATAAACTGAAATTTGATAAACTGTACAAAATG

[0299] CTGACAGAAATTTACACCGAAGATAACTTTGTGAACTTTTTTAAAGTGATTAACCGTAAAACCTATCT

[0300] GAATTTTGACAAAGCGGTGTTTCGCATTAACATTGTGCCGGATGAAAACTACACCATCAAAGATGG

[0301] CTTTAATCTGAAAGGCGCGAACCTGAGCACCAATTTTAACGGCCAGAATACCGAAATTAACAGCCG

[0302] CAATTTCACCCGTCTGAAAAATTTCACCGGCCTGTTTGAATTTTATAAACTGCTGTGCGTACGCGGC

[0303] ATTATCCCGTTTAAAACCAAAAGCCTGGATGAATAA

[0304] SEQ ID NO:19 Sumo3-HC-E1156K:LC

[0305] MGHHHHHHGSLQEEKPKEGVKTENDHINLKVAGQDGSVVQFKIKRHTPLSKLMKAYCERQGLSMRQI

[0306] RFRFDGQPINETDTPAQLEMEDEDTIDVFQQQTGGALNDLCIKVNNWDLFFSPSEDNFTNDLDKVEEIT

[0307] ADTNIEAAEENISLDLIQQYYLTFDFDNEPENISIENLSSDIIGQLEPMPNIERFPNGKKYELDKYTMFHYL

[0308] RAQEFEHGDSRIILTNSAEEALLKPNVAYTFFSSKYVKKINKAVEAFMFLNWAEELVYDFTDETNEVTTM

[0309] DKIADITIIVPYIGPALNIGNMLSKGEFVEAIIFTGVVAMLEFIPEYALPVFGTFAIVSYIANKVLTVQTINNAL

[0310] SKRNEKWDEVYKYTVTNWLAKVNTQIDLIREKMKKALENQAEATKAIINYQYNQYTEEEKNNINFNIDDL

[0311] SSKLNESINSAMININKFLDQCSVSYLMNSMIPYAVKRLKDFDASVRDVLLKYIYDNRGTLVLQVDRLKD

[0312] EVNNTLSADIPFQLSKYVDNKKLLSTFTEYIKNIVNTSILSIVYKKDDLIDLSRYGAKINIGDRVYYDSIDKN

[0313] QIKLINLESSTIEVILKNAIVYNSMYENFSTSFWIKIPKYFSKINLNNEYTIINCIENNSGWKVSLNYGEIIWT

[0314] LQDNKQNIQRVVFKYSQMVNISDYINRWIFVTITNNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLD

[0315] GCRDPRRYIMIKYFNLFDKELNEKEIKDLYDSQSNSGILKDFWGNYLQYDKPYYMLNLFDPNKYVDVNN

[0316] IGIRGYMYLKGPRGSVVTTNIYLNSTLYKGTKFIIKKYASGNEDNIVRNNDRVYINVVVKNKEYRLATNAS

[0317] QAGVEKILSALEIPDVGNLSQVVVMKSKDDQGIRNKCKMNLQDNNGNDIGFIGFHLYDNIAKLVASNWYNRQVGKASRTFGCSWEFIPVDDGWGESSL*

[0318] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVP

[0319] VSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHDVLNLTRNGY*

[0320] SEQ ID NO:20 Sumo3-HC-E1156K:LC DNA sequence

[0321] ATGGGTCATCACCATCATCATCACGGGTCCCTGCAGGAGGAGAAGCCCAAGGAGGGTGTGAAGA

[0322] CAGAGAATGACCACATCAACCTGAAGGTGGCCGGGGCAGGACGGCTCCGTGGTGCAGTTCAAGAT

[0323] CAAGAGGCACACGCCGCTGAGCAAGCTGATGAAGGCCTACTGCGAGAGGCAGGGCTTGTCAATG

[0324] AGGCAGATCAGATTCAGGTTCGACGGGCAGCCAATCAATGAAACTGACACTCCAGCACAGCTGGA

[0325] GATGGAGGACGAGGACACCATCGACGTGTTCCAGCAGCAGACGGGAGGAGCGCTGAATGATTTA

[0326] TGCATTAAAGTTAACAACTGGGATCTGTTTTTAGCCCGAGCGAAGATAACTTTACCAATGACCTGG

[0327] ATAAAGTGGAAGAAATTACCGCGGAATACCAATATTGAAGCGGCGGAAGAAAACATTAGCCTGGATC

[0328] TGATTCAACAGTACTACCTGACCTTTGATTTTGATAATGAACCGGAAACATTAGTATTGAAAACCT

[0329] GAGCAGCGATATTATTGGTCAGCTGGAACCGATGCCGAATATCGAACGCTTTCCGAACGGCAAAA

[0330] AATATGAACTGGATAAATATACGATGTTCCATTACCTGCGCGCGCAGGAATTTGAACATGGCGATT

[0331] CGCGCATTATTCTGACCAACAGCGCGGAAGAGGCGCTGCTGAAACCGAATGTGGCCTACACCTTC

[0332] TTCAGCAGCAAATACGTCAAAAAAATTAATAAAGCGGTGGAAGCGTTTATGTTCTGAACTGGGCG

[0333] GAAGAGCTGGTATACGATTTTACCGATGAAACCAACGAAGTGACCACTATGGATAAAATTGCAGAC

[0334] ATCACCATTATTGTGCCGTATATTGGCCCGGCTCTGAATATTGGCAATATGCTGAGCAAAGGCGAA

[0335] TTTGTGGAAGCCATTATTTTTACCGGCGTGGTTGCGATGCTGGAATTTATTCCGGAATATGCCCTG

[0336] CCGGTGTTCGGCACCTTTGCGATTGTGAGCTACATCGCCAACAAAGTTCTGACCGTGCAGACCATT

[0337] AACAATGCGCTGAGCAAACGCAATGAAAAATGGGATGAAGTGTACAAATATACGGTAACTAATTGG

[0338] CTGGCGAAAGTTAACACCCAGATCGATCTGATTCGCGAAAAAATGAAAAAAGCGCTGGAAAACCA

[0339] GGCGGAAGCGACCAAGGCGATCATTAACTACCAGTACAACCAGTATACCGAAGAAGAAAAAAACA

[0340] ATATCAATTTTAATATCGATGATCTGAGCAGCAAACTGAACGAATCAATTAACAGCGCGATGATTAA

[0341] CATTAACAAATTTCTGGATCAGTGCAGCGTTAGTTATCTGATGAACAGCATGATTCCGTACGCCGT

[0342] GAAACGCCTGAAAGATTTCGATGCGTCGGTGCGCGATGTGCTGCTGAAATATATCTACGATAATCG

[0343] TGGCACCCTGGTGCTGCAGGTGGATCGCTTAAAAGATGAAGTGAACAATACCCTGAGCGCGGATA

[0344] TTCCGTTTCAGCTGTCGAAATACGTGGATAACAAGAAACTGCTGAGCACCTTTACCGAATATATCAA

[0345] AAATATTGTGAACACCAGCATTCTGAGCATCGTGTACAAAAAAGATGATCTGATTGATCTGAGCCG

[0346] CTATGGTGCCAAAATTAATATTGGTGATCGTGTGTACTACGATTCTATTGATAAAAATCAGATTAAA

[0347] CTGATTAATCTGGAAAGCTCGACCATTGAAGTGATCCTGAAAAATGCGATTGTGTATAATAGCATGT

[0348] ATGAAAACTTCAGCACCAGCTTTTGGATTAAAATTCCGAAATACTTCAGCAAAATTAATCTGAATAA

[0349] CGAATACACAATTATTAATTGCATTGAAAACAATAGCGGCTGGAAAGTGAGCCTGAACTATGGCGA

[0350] GATTATTTGGACCCTGCAGGACAACAAACAGAACATTCAGCGCGTTGTGTTTAAATATAGCCAGAT

[0351] GGTGAATATTAGCGATTATATTAATCGCTGGATCTTTGTGACGATAACCAATAACCGTCTGACCAAA

[0352] AGCAAAATTTATATTAACGGCCGTCTGATTGATCAGAAACCGATTAGCAACCTGGGCAACATTCAT

[0353] GCCAGCAACAAAATTATGTTCAAACTGGATGGCTGCCGCGATCCGCGTCGTTATATTATGATCAAA

[0354] TATTTTAACCTGTTTGATAAAGAACTGAACGAAAAAGAAATCAAAGATTTATATGATAGCCAGAGCA

[0355] ATAGCGGTATTCTGAAAGATTTCTGGGGCAATTATCTGCAGTATGATAAACCGTACTATATGCTGAA

[0356] CCTGTTTGATCCGAACAAATATGTGGATGTGAACAACATTGGCATTCGTGGCTATATGTATCTGAAA

[0357] GGTCCGCGTGGCTCGGTAGTGACCACCAACATTTACCTGAACTCGACCCTCTATAAAGGTACCAA

[0358] ATTCATTATTAAAAAATACGCGAGCGGCAACGAAGATAATATTGTGCGCAACAATGATCGTGTGTAT

[0359] ATTAACGTGGTGGTGAAAAATAAAGAATACCGCCTGGCGACGAATGCAAGCCAGGCGGGCGTGG

[0360] AAAAAATTCTGAGCGCGCTGGAAATTCCGGATGTGGGCAACCTGAGTCAGGTGGTTGTGATGAAA

[0361] TCTAAAGATGATCAGGGTATTCGCAATAAATGTAAAATGAACCTGCAGGATAATAATGGCAATGATA

[0362] TCGGCTTCATTGGTTTTCATCTGTATGACAACATTGCGAAACTGGTGGCGAGCAACTGGTATAATC

[0363] GTCAAGTGGGCAAAGCGAGCCGCACCTTTGGCTGCAGCTGGGAATTTATTCCGGTGGATGATGGC

[0364] TGGGGTGAAAGCAGCCTGTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAG

[0365] CAATAACTAGCATAACCCCTTGGGGCTCTAAACGGGTCTTGAGGGGTTTTTTGCGATCCCGCGAA

[0366] ATTAATACGACTCACTATAGGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTT

[0367] AACTTTAAGAAGGAGATATACCATGCCGTTTGTGAACAAACAGTTTAACTACAAAGATCCTGTTTAAT

[0368] GGCGTTGATATTGCGTACATTAAATTCCGAACGCCGGTCAGATGCAGCCAGTGAAAGCGTTTAAA

[0369] ATTCATAATAAAATTGGGTGATTCCGGAACGCGATACGTTTACCAACCCGGAAGAGGTGATCTG

[0370] AACCCGCCGCGGAAGCGAAGCAGGTGCCGGTGAGCTACTATGATTCGACCTACCTGAGCACGG

[0371] ATAACGAAAAGATAACTATCTGAAAGGCGTGACCAAACTGTTTGAACGCATTTACAGCACCGATCC

[0372] TGGGCCGCATGCTGCTGACCAGCATTGTTCGCGGCATTCCGTTTTGGGGTGGCAGCACGATTGAT

[0373] ACGGAACTGAAAGTGATCGATACCAATTGCATTAATGTGATTCAGCCGGATGGCTCATACCGCAGC

[0374] GAAGAACTGAACCTGGTGATTATTGGCCCGAGCGCGGATATCATTCAGTTTGAATGCAAATCTTTT

[0375] GGCCATGATGTGCTGAATCTGACCCGCAATGGCTCAGGCTCGACCCAGTATATTCGCTTTTCACCT

[0376] GATTTTACCTTTGGCTTTGAAGAGAGCCTGGAAGTGGATACCAACCCGCTGCTGGGTGCGGGCAA

[0377] ATTTGCCACCGATCCGGCCGTTACCCTGGCCCATGAACTGATTCATGCCGAACATCGTCTGTATGG

[0378] CATTGCGATTAATCCGAACCGTGTGTTTAAAGTGAATACCAACGCGTATTACGAAATGAGTGGCCT

[0379] GGAAGTGTCGTTTGAAGAACTGCGCACCTTTGGCGGCCATGATGCGAAATTTATTGATAGCCTGCA

[0380] GGAAAATGAATTTCGCCTGTACTATTATAACAAATTTAAAGATGTTGCGAGCACCCTGAATAAAGCG

[0381] AAAAGCATCATTGGCACCACCGCCAGCCTGCAGTACATGAAAAATGTGTTCAAAGAAAAATACCTG

[0382] CTGAGCGAAGATACCAGCGGCAAATTTAGCGTGGATAAACTGAAATTTGATAAACTGTACAAAATG

[0383] CTGACAGAAATTTACACCGAAGATAACTTTGTGAACTTTTTTAAAGTGATTAACCGTAAAACCTATCT

[0384] GAATTTTGACAAAGCGGTGTTTCGCATTAACATTGTGCCGGATGAAAACTACACCATCAAAGATGG

[0385] CTTTAATCTGAAAGGCGCGAACCTGAGCACCAATTTTAACGGCCAGAATACCGAAATTAACAGCCG

[0386] CAATTTCACCCGTCTGAAAAATTTCACCGGCCTGTTTGAATTTTATAAACTGCTGTGCGTACGCGGC

[0387] ATTATCCCGTTTAAAACCAAAAGCCTGGATGAATAA

[0388] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0389] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0390] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0391] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. A BoNT / A2 mutant, characterized in that, Compared to wild-type BoNT / A2, the BoNT / A2 mutant includes at least one of the following amino acid mutation sites: S954N, K1137F, S1142M, V1143Y, V1144R, T1153M, T1153Q, T1153R, E1156K, or E1156M, and the amino acid sequence of the wild-type BoNT / A2 is shown in SEQ ID NO:

1.

2. The BoNT / A2 mutant as described in claim 1, characterized in that, The BoNT / A2 mutant, relative to the wild-type BoNT / A2, includes at least one of the following amino acid mutation sites: V1143Y, V1144R, T1153M, T1153Q, or E1156K.

3. An isolated polynucleotide, characterized in that, Its encoding is the BoNT / A2 mutant as described in claim 1 or 2.

4. An expression carrier, characterized in that, The expression vector contains the isolated polynucleotide as described in claim 3.

5. An expression system, characterized in that, The expression system contains the expression vector as described in claim 4 or the genome in which exogenous polynucleotides as described in claim 3 are integrated.

6. The expression system as described in claim 5, characterized in that, The host cell of the expression system is selected from eukaryotic or prokaryotic cells; preferably, the host cell is selected from any one of Escherichia coli, Klebsiella pneumoniae, Bacteroides ovalis, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides polymorpha, Bacteroides vulgaris, Bacteroides monomorpha, Lactobacillus casei, Bacteroides fragilis, Acinetobacter rumeni, Fusobacterium nucleatum, Bacteroides johnsonii, Bacteroides thaliana, Lactobacillus rhamnosus, Bacteroides masei, Bacteroides fecalis, Fusobacterium death, or Bifidobacterium breve; more preferably, the expression system is selected from Escherichia coli.

7. A pharmaceutical composition comprising the BoNT / A2 mutant as described in claim 1 or 2, and a pharmaceutically acceptable carrier or excipient.

8. The use of the BoNT / A2 mutant of claim 1 or 2, the polynucleotide of claim 3, the expression vector of claim 4, the expression system of claim 5 or 6, or the pharmaceutical composition of claim 7 in the preparation of a drug, cosmetic product, or drug targeted delivery vector.

9. The application as described in claim 8, characterized in that, The drug is for the treatment of one or more of the following conditions: neuromuscular diseases, chronic pain, glandular secretion disorders, or urinary system dysfunction.

10. A method for expressing recombinant botulinum toxin or a mutant, characterized in that, (1) The heavy and light chains of the recombinant botulinum toxin mutant are expressed as two separate polypeptide chains in the same engineered bacterium, and then the assembly of the complete botulinum toxin is automatically completed; and (2) The amino acid sequence of the first polypeptide chain, from the N-terminus to the C-terminus, consists of a protein tag sequence (SEQ ID NO:2), a recombinant botulinum toxin, or the heavy chain of a mutant; and (3) The amino acid sequence of the second polypeptide chain is the light chain of recombinant botulinum toxin or a mutant.

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