Recombinant polypeptide and method for preparing neurotoxin

Abstract

A recombinant polypeptide and a method for producing a neurotoxin using the recombinant polypeptide.

Description

This application is a divisional application of Chinese patent application 202480004872.7, with the invention name “A recombinant polypeptide and a method for preparing neurotoxin” and the application date of October 14, 2024. Cross-references to related applications

[0001] This application claims priority to the Chinese patent application filed with the State Intellectual Property Office of China on October 13, 2023, with application number 202311328450.9 and invention name “Polypeptides for efficient and rapid recombinant expression of neurotoxin proteins and methods thereof”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the field of neurotoxins, and specifically relates to a recombinant polypeptide comprising a light chain of a neurotoxin and / or a heavy chain of a neurotoxin and a connecting peptide, and a method for preparing a neurotoxin by recombinantly expressing the polypeptide. Background Art

[0003] Neurotoxins are chemicals that primarily act on ion channels. These toxins can target the junctions between motor nerves and muscles, preventing skeletal muscle contraction. In severe cases, this can lead to respiratory paralysis and death from asphyxiation. Certain organisms in nature also contain or can release these toxins, such as botulinum neurotoxin (BoNT), tetanus neurotoxin (TeNT), and diphtheria toxin.

[0004] Botulinum neurotoxins (BoNTs), also known as botulinum toxins or botulinum toxins (BTs), are neurotoxin proteins produced by the Gram-positive bacterium Clostridium botulinum during its reproduction. They are 10,000 times more toxic than potassium cyanide, with a minimum lethal dose of approximately 0.1 μg for humans. Based on their toxicity and protein antigenicity, botulinum neurotoxins are classified into seven major serotypes (BoNT / A to BoNT / G): A, B, C, D, E, F, and G. Botulinum toxin type A (also known as botulinum toxin A), produced by type A botulinum toxin, is the most toxic; types C and D toxins primarily cause poisoning in livestock, poultry, and wild birds.

[0005] The structures of all BoNT serotypes are essentially identical, consisting of a disulfide-linked double-chain structure consisting of two subunits: a light chain (LC) and a heavy chain (HC). The LC is a 50-kDa zinc metalloprotease and serves as the active component of BoNT. The HC, with a molecular weight of 100 kDa, consists of an N-terminal translocation domain (HN) and a C-terminal cell-binding domain (HC), serving as the transporter for BoNT. Other neurotoxins, such as tetanus neurotoxin and diphtheria toxin, also have a disulfide-linked double-chain structure, with a light chain and a heavy chain, respectively.

[0006] BoNTs are known for their ability to induce muscle relaxation and paralysis. BoNT / A, in particular, has been used in a variety of medical and cosmetic procedures, including the treatment of glabellar lines, hypermobility lines, migraines, hemifacial spasm, bladder hyperactivity, hyperhidrosis, nasolabial lines, cervical dystonia, blepharospasm, and spasticity. Due to its potent neurotoxicity, with a median lethal dose (LD50) of 1 ng / kg, the production and sale of botulinum toxin products are strictly regulated in various countries.

[0007] As of 2022, only two botulinum toxins, type A and type B, have been developed for commercial use. Type A is the most potent, with approximately 20 type A botulinum toxin products approved for marketing worldwide (Journal of Neural Transmission (2022) 129:829–833). Type B is primarily used for patients who develop antibodies to type A botulinum toxin. Only one type B botulinum toxin product, MyoBloc, produced by Solstice in the United States, has been approved for marketing by the US FDA (J Neurol 2003, 249:1729-1732).

[0008] Currently, all products on the market are derived from Clostridium botulinum toxin. Due to its toxicity, expression level, and purification process limitations, the production cycle of Clostridium botulinum toxin is long, the cost is high, and the production process is less safe.

[0009] Therefore, there is still a need to develop a recombinant botulinum toxin and a preparation method thereof that has high yield and purity, does not introduce additional amino acids and therefore has good safety, is generally applicable and has a simple process. Summary of the Invention

[0010] The inventors unexpectedly discovered that using a protease that can recognize and cleave the connecting peptide in the recombinant polypeptide to cleave the recombinant polypeptide used to produce the neurotoxin can obtain a complete neurotoxin without causing significant miscleavage of the enzyme cleavage sites within the light chain and heavy chain of the neurotoxin, and completed the present invention on this basis.

[0011] One of the purposes of the present invention is to address the above-mentioned problems existing in the production process of neurotoxins in the prior art and to provide a recombinant polypeptide for preparing neurotoxins and a preparation method and application thereof.

[0012] In one aspect, the present invention provides a recombinant polypeptide comprising a light chain of a neurotoxin and / or a heavy chain of a neurotoxin, and a connecting peptide;

[0013] The connecting peptide comprises a tag peptide segment and at least one or two enzyme cleavage site sequences selected from the following: a first enzyme cleavage site sequence and a second enzyme cleavage site sequence;

[0014] After the first cleavage site sequence is recognized by the protease, the protease cuts at the connection between the light chain and the connecting peptide, and / or after the second cleavage site sequence is recognized by the protease, the protease cuts at the connection between the connecting peptide and the heavy chain.

[0015] In one embodiment, the recombinant polypeptide comprises a light chain and a heavy chain of a neurotoxin and a connecting peptide located between the light chain and the heavy chain, for example, from the N-terminus to the C-terminus, it comprises a light chain of a neurotoxin, a connecting peptide and a heavy chain of a neurotoxin, so that the light chain and the heavy chain are connected into a polypeptide chain by the connecting peptide.

[0016] In another embodiment, the recombinant polypeptide comprises the light chain of the neurotoxin connected to a connecting peptide, for example, comprising the light chain of the neurotoxin and the connecting peptide from the N-terminus to the C-terminus, and / or

[0017] It comprises the heavy chain of the neurotoxin connected to a connecting peptide, for example comprising the connecting peptide and the heavy chain of the neurotoxin from the N-terminus to the C-terminus,

[0018] The connecting peptide connected to the light chain and the connecting peptide connected to the heavy chain are the same or different.

[0019] In a specific embodiment, said light chain and said heavy chain are in separate polypeptide chains.

[0020] In another embodiment, the neurotoxin is selected from botulinum neurotoxin, tetanus neurotoxin, and diphtheria toxin. For example, the botulinum neurotoxin is selected from serotypes A, B, C, D, E, F, and G, preferably from serotypes A, B, and E, such as serotype A botulinum neurotoxin.

[0021] In a specific embodiment, the amino acid sequence of the light chain is the amino acid sequence shown in SEQ ID NO: 1, and / or the amino acid sequence of the heavy chain is the amino acid sequence shown in SEQ ID NO: 2. The light chain and / or heavy chain may also be sequence variants having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO: 1 or 2 and retaining botulinum toxin activity, and these sequence variants may be naturally occurring or artificially engineered.

[0022] In a specific embodiment, the first restriction enzyme cleavage site sequence and the second restriction enzyme cleavage site sequence are independently selected from the restriction enzyme cleavage site sequences of Kex2 enzyme, EK enzyme, and TEV enzyme, for example, wherein the first restriction enzyme cleavage site sequence is selected from R, K, RR, KK, RK and KR, and / or the second restriction enzyme cleavage site sequence is selected from R, K, RR, KK, RK, KR and DDDDK.

[0023] In a specific embodiment, the first restriction enzyme cleavage site sequence and the second restriction enzyme cleavage site sequence are both restriction enzyme cleavage site sequences of Kex2 enzyme.

[0024] In another embodiment, the tag peptide segment is selected from a polyhistidine tag (poly(His)-tag), a FLAG tag (FLAG-tag), a streptavidin binding tag (Strep-tag), and a glutathione S-transferase tag (GST-tag). In a specific embodiment, the tag peptide segment is selected from a polyhistidine tag (poly(His)-tag), such as 4, 5, 6, 7, 8, 9, or 10 polyhistidines.

[0025] In another embodiment, the recombinant polypeptide further comprises a first auxiliary sequence, wherein the first linker sequence is located between the first restriction enzyme cleavage site sequence and the tag peptide segment. Furthermore, the recombinant polypeptide may further comprise a second auxiliary sequence, wherein the second auxiliary sequence is located between the second restriction enzyme cleavage site sequence and the tag peptide segment.

[0026] In a specific embodiment, the first and second helper sequences are each independently at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length.

[0027] In another specific embodiment, the first auxiliary sequence and the second auxiliary sequence are each independently selected from GSGS, EEGSGS, GSGSDDGSGS, GSGSEDGSGS, GSGSDEGSGS, EEAE, DDAD, TEEAEKL, TDDADKL, TEDAEKL, TDEADKL, GTEEAEKLG, EGTEEAEKLG, EGTDDADKLG, EGTEEADKLG, EGTDDAEKLG or EGTEDADKLG and EGTDEAEKLG, and sequences having at least 60%, 70%, 80%, or 90% identity to any of the above sequences.

[0028] In a specific embodiment, the amino acid sequence of the recombinant polypeptide is selected from SEQ ID NO:4, SEQ ID NO:15 and SEQ ID NO:19.

[0029] In a second aspect, the present invention provides an isolated polynucleotide encoding the recombinant polypeptide according to the first aspect of the present invention.

[0030] In a specific embodiment, the nucleotide sequence of the polynucleotide is selected from SEQ ID NO:5, SEQ ID NO:16 and SEQ ID NO:20.

[0031] In a third aspect, the present invention provides an expression vector comprising the polynucleotide according to the second aspect of the present invention.

[0032] In a fourth aspect, the present invention provides a cell comprising the polynucleotide according to the second aspect or the expression vector according to the third aspect of the present invention.

[0033] In one embodiment, the cell is selected from prokaryotic cells and eukaryotic cells; for example, the prokaryotic cell is selected from Escherichia coli cells; for example, the eukaryotic cell is selected from yeast cells, insect cells, plant cells, and mammalian cells.

[0034] In a fifth aspect, the present invention provides a method for producing a neurotoxin, comprising culturing the cell described in the fourth aspect above under conditions suitable for expressing the recombinant polypeptide.

[0035] In one embodiment, the method further comprises isolating the recombinantly expressed recombinant polypeptide and cleaving the recombinant polypeptide with a protease.

[0036] In another embodiment, the method further comprises purifying the neurotoxin from the cell culture.

[0037] In a sixth aspect, the present invention provides a pharmaceutical or cosmetic composition comprising the neurotoxin produced according to the method of the fifth aspect above.

[0038] In a seventh aspect, the present invention provides the use of the recombinant polypeptide described in the first aspect, the polynucleotide described in the second aspect, the expression vector described in the third aspect, or the cell described in the fourth aspect in the preparation of a neurotoxin, for example, the neurotoxin is selected from tetanus neurotoxin, diphtheria toxin and botulinum neurotoxin.

[0039] After the recombinant polypeptide of the present invention is used for the fusion expression of neurotoxins, the target neurotoxin protein with biological activity can be prepared quickly and in large quantities. The recombinant neurotoxin polypeptide designed by the present invention has an affinity purification tag, which is easy to prepare using the separation and purification methods commonly used in industry, is easy to operate, and has low production costs. The affinity tag used for purification is combined with the enzyme cleavage site of the protease, so that the protease selectively acts on the polypeptide of the present invention to perform enzymatic cleavage, avoiding the enzymatic cleavage of the neurotoxin protein sequence, which not only improves the enzymatic cleavage efficiency of the fusion expression protein, but also improves the integrity of the fusion expression neurotoxin protein, and increases protein yield and activity. In addition, after enzymatic cleavage, the neurotoxin produced by the obtained fusion expression no longer contains exogenous amino acids or polypeptides, thereby improving safety.

[0040] On the other hand, the recombinant polypeptide constructed by the present invention, for example, does not contain the native linker sequence between the light and heavy chains. This, on the one hand, ensures that the expressed recombinant neurotoxin protein is non-toxic before the enzymatic cleavage step, greatly improving the safety of the production process. Furthermore, it avoids the disordered cleavage of the native linker sequence by cellular proteases, significantly reducing protein polymorphism, improving the homogeneity of the produced neurotoxin protein, and facilitating subsequent protein purification, significantly improving the yield, purity, and toxicity of the target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The figure is a schematic diagram of an exemplary recombinant botulinum toxin A protein structure and its expression vector; wherein A is a schematic diagram of the recombinant botulinum toxin A protein structure, B is the Kex2 restriction site in the recombinant botulinum toxin A protein, and C is a schematic diagram of the expression vector plasmid into which the recombinant botulinum toxin gene sequence is inserted.

[0042] Figure 2Figures 1 and 2 show the results of nickel affinity chromatography purification of recombinant botulinum toxin A and analysis of its stability to enzyme cleavage. Figure A shows the electrophoresis of the nickel affinity chromatography purification results, with the arrow indicating recombinant botulinum toxin; Figure B shows the electrophoresis of the Kex2 protease-cleaved purified sample; Figure C shows a Western blot of the enzyme cleavage results. The sample containing the HIS tag before cleavage (0 min) was loaded at 100 ng, while the sample loading for the other samples was 3 μg. In each figure, M represents the protein molecular weight marker.

[0043] Figure 3 Figure 3 shows the results of recombinant botulinum toxin A after enzymatic cleavage and a second nickel-column affinity chromatography purification. Figure A shows the reduced SDS-PAGE electrophoresis analysis after enzymatic cleavage, Figure B shows the effect of enzymatic cleavage detected by Western blot (the sample loading amount before enzymatic cleavage for the HIS-tagged protein was 30 ng, and the sample loading amount for other samples was 3 μg), and Figure C shows the reduced SDS-PAGE electrophoresis after nickel-column affinity chromatography after enzymatic cleavage. Figure D shows the electrophoresis of the recombinant protein containing the natural botulinum toxin sequence 438-448 (treated at room temperature for 3 h and then run on SDS-PAGE). In each figure, M is the protein molecular weight marker.

[0044] Figure 4 Figures 2 and 3 are the results of anion HQ column chromatography purification; A is the electrophoresis diagram of the HQ column separation and purification results; B is the electrophoresis diagram of the Superdex 200 column separation and purification results. In each figure, M is the protein molecular weight marker.

[0045] Figure 5 The purity and HIS residue test results of the prepared recombinant botulinum toxin A are shown in Figure 1; A is an SDA-PAGE electrophoresis diagram, B is a Western blot analysis diagram (the "positive" sample is a purified, non-enzymatically digested, HIS-tagged recombinant botulinum toxin protein), and C is an HPLC chromatogram. In each figure, M is the protein molecular weight marker.

[0046] Figure 6 The primary structure detection results of recombinant botulinum toxin A prepared for mass spectrometry analysis; among them, A is the protein's precise molecular weight detection result diagram, B is the C430 / 454 disulfide bond position detection result diagram, C is the C1235 / 1280 disulfide bond position detection result diagram, and D is the protein's N- and C-terminal amino acid sequence detection result diagram.

[0047] Figure 7 These are the toxicity test results of the recombinant botulinum toxin A prepared in the present invention; wherein, A is the toxicity result of the recombinant botulinum toxin protein A injected through the tail vein; B is the toxicity result of the recombinant botulinum toxin protein A injected through the intraperitoneal method; C is the toxicity test result of the recombinant botulinum toxin A protein activated with various auxiliary sequences.

[0048] Figure 8The present invention is a Kex2 protease cleavage site in the recombinant botulinum toxin B protein for preparing recombinant botulinum toxin B.

[0049] Figure 9 Figure 1 shows the results of the first nickel affinity chromatography and anion exchange purification of recombinant botulinum toxin B. A is the electrophoretogram of the first nickel affinity chromatography; B is the electrophoretogram of the first anion HQ column chromatography.

[0050] Figure 10 Figures show the results of recombinant botulinum toxin B digestion and secondary nickel affinity chromatography purification. A shows an SDS-PAGE electrophoresis of samples digested with Kex2 protease and purified by secondary nickel affinity chromatography; B shows a Western blot image. The sample containing the HIS tag before digestion (0 min) was loaded at 100 ng, while the other samples were loaded at 3 μg. In each figure, M represents the protein molecular weight marker.

[0051] Figure 11 Figures 1 and 2 show the results of the second purification of recombinant botulinum toxin B by anion column and molecular sieve chromatography. Figure A shows the electropherogram of separation and purification using an HQ column; Figure B shows the electropherogram of separation and purification using a Superdex 200 column; and Figure C shows the reduced SDA-PAGE electropherogram of the prepared recombinant botulinum toxin B. In each figure, M represents the protein molecular weight marker.

[0052] Figure 12 The present invention is for preparing recombinant botulinum toxin E protein Kex2 enzyme cleavage site.

[0053] Figure 13 Figure 2 shows the results of nickel affinity chromatography purification of recombinant botulinum toxin E. M represents the protein molecular weight marker.

[0054] Figure 14 Results of recombinant botulinum toxin E after enzymatic cleavage and a second nickel-column affinity chromatography purification; A is the SDS-PAGE electrophoresis of nickel-column affinity chromatography after enzymatic cleavage, B is the Western blot analysis of the enzymatic cleavage effect (the sample loading amount before enzymatic cleavage for HIS-tagged proteins is 30 ng, and the sample loading amount for other samples is 3 μg). M is the protein molecular weight marker.

[0055] Figure 15 Figure 1 shows the results of purification of recombinant botulinum toxin E by anion column and molecular sieve chromatography; A is the electrophoresis diagram of the separation and purification results of HQ column; B is the electrophoresis diagram of the separation and purification results of Superdex 200 column; in each figure, M is the protein molecular weight marker.

[0056] Figure 16Figures showing the purity and HIS residue detection results of the prepared recombinant botulinum toxin E; A is an SDA-PAGE electrophoresis diagram, and B is a Western blot analysis diagram (the "positive" sample is the purified, undigested, HIS-tagged recombinant botulinum toxin protein). In each figure, M is the protein molecular weight marker. DETAILED DESCRIPTION definition

[0057] Unless otherwise defined, the meaning of the scientific and technical terms used herein is the meaning commonly understood by those skilled in the art. The nomenclature and techniques used in cell and tissue culture, molecular biology, protein and oligo- or polynucleotide chemistry and hybridization described herein are well known in the art and commonly used. For recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (such as electroporation, lipofection), standard techniques are used. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions or the methods commonly used in this area or as described herein. The aforementioned techniques and methods are usually used as described in the multiple comprehensive and more specific documents that are well known in the art and quoted and discussed in this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)). The nomenclature and laboratory methods and techniques used in analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well known in the art and commonly used.

[0058] As used herein, "neurotoxin" refers to a toxic substance that is destructive to nervous tissue. It can be a natural neurotoxin or a recombinant neurotoxin, as long as it has neurotoxin activity. The light chain / heavy chain of a neurotoxin refers to the different polypeptide chains of the neurotoxin connected by disulfide bonds. Generally, the light chain refers to the polypeptide chain with toxic activity, and the heavy chain refers to the polypeptide chain with binding activity.

[0059] As used herein, "connecting peptide" refers to an exogenous amino acid sequence that is used to connect a light chain or a heavy chain, or to connect both the light chain and the heavy chain so that the light chain and the heavy chain are located on the same polypeptide chain. The connecting peptide comprises an affinity tag sequence and at least one enzyme cleavage site sequence selected from the following: a first enzyme cleavage site sequence and a second enzyme cleavage site sequence. As used herein, "enzyme cleavage site sequence" refers to a sequence that can be recognized by a protease (also called a recognition site), in which there is a specific cleavage site in or next to the sequence, and the protease cuts the polypeptide chain at the cleavage site. As used herein, "cutting at the junction" means that after the protease cuts, the connecting peptide is completely removed from the light chain and / or heavy chain.

[0060] Kex2 enzyme, also known as Kex2 protease, recombinant two-base endonuclease, and YSCF protease, can specifically recognize and cleave the carboxyl-terminal peptide bonds of dibasic amino acids such as Arg-Arg, Lys-Arg, and Pro-Arg. Its recognition sequences include RR, KK, RK, or KR.

[0061] EK enzyme, also known as enterokinase, is a type of serine protease that specifically recognizes and cleaves the DDDDK sequence, and its recognition sequence includes DDDDK.

[0062] TEV enzyme is a protease derived from tobacco etch virus (TEV). It can specifically recognize the heptapeptide sequence EXXYXQG / S and cleave between Gln and Gly / Ser amino acid residues. Its commonly used recognition sequences include ENLYFQG or ENLYFQS.

[0063] As used herein, a "tag peptide" refers to a sequence added to a polypeptide chain for the purpose of separation and purification, which utilizes affinity adsorption and dissociation between biomolecules to achieve the purpose of protein purification.

[0064] A poly(His)-tag refers to an amino acid sequence composed of four, five, or six or more consecutive histidines, such as HHHHHH, HHHHHHH, or HHHHHHHHH.

[0065] The FLAG tag (FLAG-tag) is a short, hydrophilic, 8-amino acid peptide (DYKDDDDK). The streptavidin-binding tag (Strep-tag) is a short peptide tag that specifically binds to streptavidin and is divided into Strep-tag and Strep-tag II. The glutathione S-transferase tag (GST-tag) is a recombinant GST enzyme that specifically binds to glutathione. These tag peptides with specific affinity can be used to specifically isolate peptides carrying the tag.

[0066] The first auxiliary sequence and the second auxiliary sequence, in this article, refer to the sequences located between the first enzyme cleavage site sequence (the second enzyme cleavage site sequence) and the tag peptide segment. Setting the auxiliary sequences can help the enzyme cleavage site sequence, the tag peptide segment and the prepared recombinant neurotoxin to better perform their respective functions.

[0067] As an example, this application involves the following technical solutions:

[0068] 1. A polypeptide for recombinant expression to produce neurotoxin protein, characterized in that

[0069] Tag peptides, which are used for affinity purification;

[0070] The first enzyme cleavage site, which includes at least one amino acid and is located at the N-terminal side of the tag peptide segment;

[0071] The second enzyme cleavage site includes at least one amino acid and is located at the C-terminal side of the tag peptide segment.

[0072] 2. The polypeptide according to embodiment 1 is characterized in that the tag peptide segment has a His-(Xm)n-His sequence, where m and n are selected from natural numbers.

[0073] 3. The polypeptide according to embodiment 2, characterized in that m and n are each selected from natural numbers 1 to 10.

[0074] 4. The polypeptide according to embodiment 2, characterized in that each of the Xm is independently selected from Lys, Met, Asp, Arg, Tyr, Glu and His.

[0075] 5. The polypeptide according to embodiment 2, characterized in that the tag peptide is selected from HHHHHH, HHHHHHH or HHHHHHHH.

[0076] 6. The polypeptide according to embodiment 1 is characterized in that the amino acid of the first cleavage site is selected from at least one of arginine and lysine.

[0077] 7. The polypeptide according to embodiment 1, characterized in that the first restriction enzyme cleavage site is selected from R, K, RR, KK, RK or KR.

[0078] 8. The polypeptide according to embodiment 1, characterized in that the amino acids at the second cleavage site include at least one arginine or lysine.

[0079] 9. The polypeptide according to embodiment 1, characterized in that the second restriction enzyme cleavage site is selected from R, K, RR, KK, RK, KR or DDDDK.

[0080] 10. The polypeptide according to embodiment 1, characterized in that the first restriction enzyme cleavage site is a Kex2 restriction enzyme cleavage site.

[0081] 11. The polypeptide according to embodiment 1, characterized in that the first restriction enzyme cleavage site and the second restriction enzyme cleavage site are Kex2 restriction enzyme cleavage sites.

[0082] 12. The polypeptide according to embodiment 1 is characterized in that the tag peptide segment further comprises a flexible sequence located at one or more of the N-terminus and the C-terminus of the tag peptide segment.

[0083] 13. The polypeptide according to embodiment 1, characterized in that the flexible sequence comprises at least one glycine or serine.

[0084] 14. The polypeptide according to embodiment 13, characterized in that the flexible sequence is selected from G, S, GS, SG, GG, SS, GGS, SGG, GSG and SGS.

[0085] 15. The polypeptide according to embodiment 13, characterized in that the flexible sequence is located between the tag peptide segment and the first enzyme cleavage site.

[0086] 16. The polypeptide according to embodiment 13, characterized in that the flexible sequence is located between the tag peptide segment and the second enzyme cleavage site.

[0087] 17. The polypeptide according to embodiment 1 is characterized in that it also includes a first auxiliary sequence, and the first auxiliary sequence is located between the tag peptide segment and the first enzyme cleavage site or between the tag peptide segment and the second enzyme cleavage site.

[0088] 18. A polypeptide according to embodiment 17, characterized in that the first auxiliary sequence is selected from GSGS, EEGSGS, GSGSDDGSGS, GSGSEDGSGS, GSGSDEGSGS, EEAE, DDAD, TEEAEKL, TDDADKL, TEDAEKL, TDEADKL, GTEEAEKLG, EGTEEAEKLG, EGTDDADKLG, EGTEEADKLG, EGTDDAEKLG or EGTEDADKLG and EGTDEAEKLG and homologous sequences thereof with a homology greater than 70%.

[0089] 19. The polypeptide according to embodiment 1, further comprising:

[0090] A first auxiliary sequence, which is located between the tag peptide segment and the first enzyme cleavage site; and

[0091] The second auxiliary sequence is located between the tag peptide segment and the second enzyme cleavage site.

[0092] 20. A polypeptide according to embodiment 19, characterized in that the first auxiliary sequence and the second auxiliary sequence are independently selected from GSGS, EEGSGS, GSGSDDGSGS, GSGSEDGSGS, GSGSDEGSGS, EEAE, DDAD, TEEAEKL, TDDADKL, TEDAEKL, TDEADKL, GTEEAEKLG, EGTEEAEKLG, EGTDDADKLG, EGTEEADKLG, EGTDDAEKLG or EGTEDADKLG and EGTDEAEKLG and homologous sequences thereof with a homology greater than 70%.

[0093] 21. The polypeptide according to embodiment 1 is characterized in that the polypeptide is used to connect one heavy chain and one light chain that constitute a neurotoxin, the light chain is connected to one end of the polypeptide, and the heavy chain is connected to the other end of the polypeptide.

[0094] 22. The polypeptide according to embodiment 1 is characterized in that the polypeptide is used to connect one heavy chain and one light chain that constitute a neurotoxin, the light chain is connected to the N-terminus of the polypeptide, and the heavy chain is connected to the C-terminus of the polypeptide.

[0095] 23. The polypeptide according to embodiment 1 is characterized in that it is used for fusion expression of a neurotoxin, wherein the light chain of the neurotoxin is connected to the N-terminus of the polypeptide, and the heavy chain is connected to the C-terminus of the polypeptide.

[0096] 24. The polypeptide according to embodiment 1, characterized in that the fusion expression is implemented in various expression systems, and the expression system is selected from a prokaryotic expression system, a eukaryotic expression system, an insect rod expression system or a mammalian expression system.

[0097] 25. The polypeptide according to embodiment 1, characterized in that the prokaryotic organism is Escherichia coli.

[0098] 26. The polypeptide according to embodiment 25, characterized in that the Escherichia coli is selected from BL21(DE3), HMS174(DE3), pLysS or pLysE.

[0099] 27. The polypeptide according to embodiment 1, characterized in that the polypeptide is used to connect one heavy chain and one light chain constituting a botulinum neurotoxin, wherein the light chain of the botulinum neurotoxin is connected to the N-terminus of the polypeptide and the heavy chain is connected to the C-terminus of the polypeptide; or

[0100] The polypeptide is used to connect a heavy chain and a light chain constituting tetanus neurotoxin, wherein the light chain of the tetanus neurotoxin is connected to the N-terminus of the polypeptide, and the heavy chain is connected to the C-terminus of the polypeptide.

[0101] 28. The polypeptide according to embodiment 27 is characterized in that the tag peptide is selected from HHHHHH, HHHHHHH or HHHHHHHH; the first enzyme cleavage site is a Kex2 enzyme cleavage site; and the second enzyme cleavage site is a Kex2 enzyme cleavage site or an enterokinase enzyme cleavage site.

[0102] 29. The polypeptide according to embodiment 28, characterized in that the amino acid sequence of the first restriction enzyme cleavage site is selected from R, K, RR, KK, RK or KR.

[0103] 30. The polypeptide according to embodiment 28, characterized in that the amino acid sequence of the second restriction enzyme cleavage site is selected from DDDDK, R, K, RR, KK, RK or KR.

[0104] 31. The polypeptide according to embodiment 27 is characterized in that it also includes a flexible sequence located at one or more of the N-terminus and C-terminus of the tag peptide segment, selected from G, S, GS, SG, GG, SS, GGS, SGG, GSG or SGS.

[0105] 32. The polypeptide according to embodiment 27 is characterized in that it also includes an auxiliary sequence located between the tag peptide segment and the first enzyme cleavage site, and at least one between the tag peptide segment and the second enzyme cleavage site.

[0106] 33. A polypeptide according to embodiment 27, characterized in that the auxiliary sequence is selected from GSGS, EEGSGS, GSGSDDGSGS, GSGSEDGSGS, GSGSDEGSGS, EEAE, DDAD, TEEAEKL, TDDADKL, TEDAEKL, TDEADKL, GTEEAEKLG, EGTEEAEKLG, EGTDDADKLG, EGTEEADKLG, EGTDDAEKLG or EGTEDADKLG and EGTDEAEKLG and homologous sequences thereof with a homology of greater than 70%.

[0107] 34. A method for producing a neurotoxin protein by recombinant expression, characterized in that the light chain and heavy chain of the neurotoxin are respectively linked to the two ends of an artificial polypeptide sequence to form a fusion expression sequence, and the fusion expression is carried out in an expression system. The protein expressed by the expression system is subjected to enzymatic digestion and separation and purification to produce a protein identical to the natural neurotoxin sequence or with greater than 70% homology;

[0108] The artificial polypeptide includes the polypeptide described in embodiment 1.

[0109] 35. The method for producing neurotoxin protein by recombinant expression according to embodiment 34 is characterized in that the expression system is selected from a prokaryotic expression system, a eukaryotic expression system, an insect rod expression system or a mammalian expression system.

[0110] 36. The method for producing neurotoxin protein by recombinant expression according to embodiment 34 is characterized in that the expression system is Escherichia coli.

[0111] 37. The method for producing neurotoxin protein by recombinant expression according to embodiment 34 is characterized in that the first restriction site of the polypeptide is a dibasic amino acid endopeptidase restriction site, and the tag peptide segment is selected from FLAG, Myc, STREP, HA or has a His-(Xm)n-His sequence, wherein m and n are respectively selected from natural numbers. Example

[0112] The technical solution of the present invention is further described in detail below in conjunction with the embodiments, but these embodiments should not be construed as limiting the present invention. Example 1 Construction of recombinant botulinum toxin A protein and engineered bacteria

[0113] Figure 1 Figure A shows the structure of an exemplary recombinant botulinum toxin A protein of the present invention. Between the light chain and the heavy chain, there is a first restriction enzyme cleavage site, a first auxiliary sequence, a tag peptide, a second auxiliary sequence, and a second restriction enzyme cleavage site. The connecting peptide used in this example to connect the light chain (SEQ ID NO: 1) and the heavy chain (SEQ ID NO: 2) of botulinum toxin A has its N-terminus connected to the light chain and its C-terminus connected to the heavy chain. Its structure from N-terminus to C-terminus is as follows:

[0114] Kex2 protease cleavage site (KR)—first auxiliary sequence (SEQ ID NO: 3)—tag peptide (consisting of 8 histidines)—second auxiliary sequence (SEQ ID NO: 3)—Kex2 protease cleavage site (KR).

[0115] The recombinant polypeptide prepared in this example (its amino acid sequence is shown in SEQ ID NO: 4) has 9 Kex2 protease cleavage sites, 7 of which are also present in the natural protein sequence of botulinum toxin, such as Figure 1 As shown in B.

[0116] The nucleotide coding sequence of the recombinant botulinum toxin amino acid sequence was designed and optimized based on the characteristics of the host Escherichia coli, as shown in SEQ ID NO: 5. The coding sequence was inserted into the pET-28 plasmid to obtain the expression vector pET-28-RDS, as shown in Figure 1 C. After sequence verification, the host bacteria E. coli BL-21 (DE3) (purchased from Thermo Fisher Scientific, USA) were transformed. Example 2 Preparation of active recombinant botulinum toxin A protein

[0117] The engineered bacteria obtained in Example 1 were cultured in shake flasks and induced to express recombinant botulinum toxin using 0.3 mM IPTG. The fermentation broth was centrifuged to obtain bacterial cells, which were then disrupted by ultrasonication. Preliminary purification of recombinant botulinum toxin protein A was achieved using nickel affinity chromatography. Non-native botulinum toxin amino acid sequences were removed from the resulting protein using two proteases, Kex2 and carboxypeptidase B. Subsequently, various separation methods, including ion exchange chromatography and molecular sieve chromatography, were used to obtain a highly purified target protein containing only the native botulinum toxin A amino acid sequence, with a purity exceeding 99%. The specific separation and purification process is as follows:

[0118] (1) Bacteria disruption

[0119] The cells were resuspended in 3 times the bacterial volume of NaCl (20 g / L) and centrifuged at 4 ° C and 8000 rpm for 10 min to collect the cells. After obtaining the cells, 9 times the bacterial volume of buffer (50 mM phosphate, 0.1 M NaCl, pH = 7.0) was added to resuspend the cells and 5‰ PMSF (100 mM) was added. Under low temperature conditions, the cells were broken using a high-pressure homogenizer (Shanghai Hongli Technology Biological Co., Ltd., 850 bar, 2 times). The lysate was centrifuged at 12,000 rpm for 40 min, the supernatant was collected, and the sample solution was filtered through a 0.45 μm filter to obtain the sample solution.

[0120] (2) Nickel affinity chromatography

[0121] In a chromatography cabinet at 4°C, the sample solution was passed through a pre-equilibrated nickel affinity chromatography column (Biyuntian Biotechnology Co., Ltd.), and then washed with 5 column volumes of equilibrium buffer (50mM phosphate, 0.1MNaCl, pH=7.0) until no protein solution flowed out. Then, a step-by-step gradient elution was used. The eluents were solutions containing 20, 50, 200, and 500mM imidazole prepared with the equilibrium buffer (each gradient was 5 column volumes). The elution peaks of each section were collected separately, and the protein purity and content of each peak were detected by SDS-PAGE electrophoresis. The experimental results showed that the recombinant botulinum toxin protein was mainly present in the 50mM imidazole eluate, and the purity was relatively high. Figure 2 A. The 50mM imidazole eluate was collected and ultrafiltered 100 times with 50mM phosphate buffer (pH=7.0). Kex2 protease was added at a ratio of 1:500 (enzyme:protein, w / w). The mixture was incubated for 5 hours. Samples were taken regularly for electrophoresis to detect the non-specific enzymatic cleavage effect of the protease on the target protein. The inventors unexpectedly discovered that after 5 hours of enzymatic cleavage, the amount of target protein did not change much, indicating that the non-specific enzymatic cleavage effect of Kex2 on the target protein was very weak. Almost no significant cleavage of the recombinant polypeptide by Kex2 at the Kex2 cleavage site present in natural botulinum toxin was observed. Figure 2B. Western blot was used to detect the effect of HIS tag removal. The results showed that as the incubation time increased, the HIS tag of the target protein gradually decreased. After 3 hours of enzyme digestion (sample loading 3μg), the reduction rate was significantly reduced, and the band intensity at this time was weaker than that of the sample before enzyme digestion (sample loading 100ng). This indicates that after 3 hours of incubation with the protease, more than 96% of the target protein was removed from the HIS tag. Figure 2 C.

[0122] It can be seen that this example has verified that the scheme of using Kex2 protease to remove exogenous polypeptides is feasible.

[0123] (3) Removal of the connecting peptide

[0124] The eluate containing a large amount of target protein after nickel column chromatography was collected and concentrated and ultrafiltered using a 50KDa ultrafiltration centrifuge tube (Sartorius). The specific operation is to concentrate the protein eluate to 1 / 6 of the original volume, then use enzyme cleavage buffer (50mM phosphate, 0.1M NaCl, pH=7.0) to dilute the protein concentrate 6 times, repeat the concentration and dilution once, and then concentrate the solution to a protein concentration of about 2mg / ml. KEX2 protease and carboxypeptidase B (Shanghai Yaxin Biotechnology Co., Ltd.) were added at a mass ratio of 1:500 (enzyme: protein, w / w). Enzyme cleavage buffer was added to make the botulinum toxin protein concentration at 0.5mg / ml, and enzyme cleavage was performed at 25°C for 3 hours. The sample after enzyme cleavage (nickel column loading solution) was subjected to reduction electrophoresis. The results showed that the protein after enzyme cleavage was mainly 100KDa and 50KDa, while the sample protein molecular weight without protease was 150KDa, indicating that the protease cleaved the target protein. It also shows that the protein before enzyme cleavage was a single chain and non-toxic, see Figure 3 A. Western blot analysis using a His tag antibody further examined the effect of HIS tag removal. The results showed that although the loading amount of the sample (3 μg) after 3 hours of digestion was 100 times that of the positive protein (30 ng), the intensity of the target band in the former was significantly lower than that in the latter, indicating that more than 99% of the HIS tag was removed. Figure 3 B.

[0125] After the enzyme digestion was completed, the enzyme digestion solution was passed through a pre-equilibrated nickel affinity chromatography column (column equilibration solution was 50 mM phosphate buffer, pH = 7.0) once at low temperature. The flow-through and wash solution (3 times the column volume) were collected, and EDTA (final concentration was 1 mM) was added. The enzyme digestion effect was detected by Western blot and SDS-PAGE. The SDS-PAGE results showed that the protein after enzyme digestion was mainly in the effluent, and there were few target protein bands in the eluate, indicating that most proteins did not carry a HIS tag. The Western blot test results also confirmed this. Figure 3 B and 3C.

[0126] The recombinant protein of this example does not carry the amino acid sequence 439-448 of natural botulinum toxin A. Our experimental results show that when the recombinant botulinum toxin A protein with this natural sequence is expressed in E. coli, it will be cleaved to form two chains even without the addition of protease, and show toxin activity. Figure 3 D. This leads to the premature appearance of toxic forms during the recombinant expression of botulinum toxin A, significantly increasing the biosafety risk of the production process.

[0127] (4) Anion exchange column chromatography

[0128] Under low temperature, the flow-through and washing liquid collected after the enzyme cleavage is completed and passed through the nickel affinity chromatography column are passed through the pre-equilibrated anion exchange column HQ (the column equilibration liquid is 50mM phosphate buffer, pH=7.0). After loading, the sample is rinsed with equilibration buffer for 5-10 column volumes, and the flow-through peak is collected; then it is eluted with a linear gradient of 0-1mol / L NaCl, with a total elution volume of 20 column volumes, and the elution peak appears after about 5 column volumes. The flow-through, washing liquid and eluate are collected, and the purity and content of the recombinant botulinum toxin protein in each sample are detected by SDS-PAGE electrophoresis. The experimental results show that the eluate is mainly impurities, and the botulinum toxin protein mainly appears in the flow-through with a purity of about 95%, see Figure 4 A. The collected flow-through was concentrated using a 50K ultrafiltration centrifuge tube (Sartorius) to a protein concentration of 5 mg / ml, and 10% glycerol was added and stored at -70°C until use.

[0129] (5) Superdex 200 gel filtration column chromatography

[0130] Under low temperature, the concentrated sample was passed through a pre-equilibrated (50 mM phosphate, 0.1 M NaCl, pH = 7.0) Superdex 200 column (Cytiva) and eluted with equilibration buffer at a flow rate of 0.5 ml / min for 2 column volumes until no peak appeared. The elution peak appeared approximately 23 minutes later. Each elution peak was collected and the protein purity and content of each peak were determined by SDS-PAGE electrophoresis. The experimental results showed that the botulinum toxin protein was mainly concentrated in elution peak I and was basically free of contaminants. Elution peaks II and III were mainly 55 kDa and 30 kDa contaminants, respectively. Figure 4 B.

[0131] (6) Sample quantification and preservation

[0132] The molecular sieve chromatography elution peak I was collected, 10% glycerol was added, and the final protein concentration was determined to be 1-2 mg / mL using the A280 and BCA methods. The purity and HIS tag residue of the final product were tested by SDS-PAGE, HPLC, and WB. The results showed that the samples showed a single band in the electrophoresis, indicating that they were electrophoretically pure. The WB results showed that the HIS tag residue was less than 1‰, and the HPLC test showed a purity of 99.19%. Figure 5 The samples were aliquoted and stored in a -70℃ refrigerator.

[0133] The inventors also tested a variety of connecting peptide structures and other affinity tags such as FLAG, all of which achieved good expression, purification and cleavage effects, and obtained recombinant botulinum toxin A protein that met the expectations of the present invention. Example 3 Sequence Identification of Recombinant Botulinum Toxin A Protein

[0134] Botulinum toxin A protein consists of two polypeptide chains, which are connected by a pair of interchain disulfide bonds. The sample was treated with or without the addition of the reducing agent TCEP (TCEP can open the interchain disulfide bonds, separate the two chains, and put the sample in a reduced state). The protein sample was centrifuged at 12000g for 30 minutes, separated by a chromatographic column ACQUITY UPLC Protein BEH 300 C4 column and entered into a Xevo G2-XS Q-TOF (Waters) mass spectrometer to determine the intact molecular weight and reduced molecular weight of the botulinum toxin A protein prepared in this example. The mass spectrometry data was analyzed after deconvolution using UNIFI software. For specific data, see Figure 6 The results showed that the measured molecular weights of the full-length, light chain, and heavy chain of the botulinum toxin A protein prepared in this example were 148049.4 Da (theoretical value 148045.3 Da), 49897.5 Da (theoretical value 49896.1 Da), and 98151.9 Da (theoretical value 98151.2 Da), respectively. The deviations from the theoretical molecular weights were all less than 28 ppm, indicating that the molecular weight of the botulinum toxin A protein prepared in this example was consistent with the theoretical value. This indicates that the amino acid sequence of the recombinant botulinum toxin A protein obtained was consistent with the designed one.

[0135] Disulfide bonds are an important form of post-translational modification of proteins. Interchain or intrachain disulfide bonds are crucial for protein molecules to maintain the correct higher-order structure and necessary biological activity. Nanoliter liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) was used to identify the intrachain and interchain disulfide bond pairing of the botulinum toxin A protein prepared in this embodiment. The nanoliter liquid chromatography and mass spectrometer were EASY-nLC 1200 and Thermo Orbitrap QExactive HF from Thermo Corporation of the United States, respectively, and the liquid analysis column was a C18 reverse phase column. Mass spectrometry data were obtained through BioPharma Finder (V5.2) software analysis shows that each pair of disulfide bonds has collected abundant primary and secondary ion information, with the primary mass deviation of the ion signal value being 10ppm and the secondary mass deviation being 20ppm. Figure 6 B). In this example, the botulinum toxin A prepared in this embodiment forms a disulfide bond between cysteine ​​429 of the light chain and cysteine ​​6 of the heavy chain (i.e., corresponding to the disulfide bond formed between cysteine ​​430 and 454 of the natural botulinum toxin protein). Also, a disulfide bond is formed between cysteine ​​787 and cysteine ​​832 of the heavy chain (i.e., corresponding to the disulfide bond formed between cysteine ​​1235 and 1280 of the natural botulinum toxin A protein). For detailed values, see Figure 6 C. The results showed that the botulinum toxin protein prepared in this example contained a pair of interchain disulfide bonds and a pair of intrachain disulfide bonds, and the disulfide bond connection mode was consistent with the theory.

[0136] The amino acid sequence is the basis for protein function. Figure 6 -A The results suggest that the amino acid sequence of the recombinant botulinum toxin A protein we obtained is the same as the designed one. In order to further verify this result, the C- and N-terminal sequences of the protein were detected. The protein sample prepared in this example was denatured with guanidine hydrochloride, reduced, and enzymatically hydrolyzed with protease Glu-C and Trypsin, and then separated by a chromatographic column ACQUITY UPLC Protein BEH 300 C4 column and entered into the Xevo G2-XS Q-TOF (Waters) mass spectrometer to determine the N-terminal and C-terminal amino acid sequences of the protein, and the mass spectrometry data were analyzed using UNIFI software. The results showed that the N-terminal and C-terminal amino acid sequences of the light chain of the botulinum toxin A protein prepared in this example were PFVNK and LLCRVGIITS, respectively, and the N-terminal and C-terminal amino acid sequences of the heavy chain were ALNDLCIK and VDDGWGERPL, respectively, which were consistent with the theoretical sequence, see Figure 6 D. This also indicates that all exogenous amino acid sequences are removed after enzyme digestion. Example 4 Toxicity Determination of Recombinant Botulinum Toxin A Protein

[0137] Five SPF Kunming mice aged 26 to 30 days were injected into the tail vein of each mouse with 0.1 ml of the recombinant botulinum toxin A product prepared in this example at a concentration of 10 μg / ml. The average time to death (in minutes) was calculated according to the formula Y = 17776578 - 868930 × t + 14382 × t 2 -78.90×t 3 The toxicity of the samples was calculated, where t is the death time in minutes and Y is the corresponding toxicity calculated by the regression equation. Three batches were tested. The results showed that the LD50 (pg / 25g, U) of the prepared recombinant botulinum toxin A was 5.48 and 5.64, respectively. The average toxicity unit of recombinant botulinum toxin A was 5.56 pg / U, and the average toxicity unit (U) per mg of product for Kunming mice was 18×10 7 ,See Figure 7 A.

[0138] Intraperitoneal injection:

[0139] Serially dilute the sample in equal doses and intraperitoneally inject 0.5 ml of each dilution into five female Kunming mice aged 26 to 30 days using a disposable sterile syringe. Observe the animals for morbidity and mortality at least once daily for four consecutive days. Record the number of deaths. Test three batches in total. Calculate the LD50 of the sample using the Reed-Muench method based on the number of deaths within four days. The formula is:

[0140] The results showed that the LD50 (pg / 25g, U) of the two measurements were 5.3 and 6.2, respectively. That is, the average toxicity unit of recombinant botulinum toxin A was 5.75 pg / U, and the number of toxicity units (U) per mg of product for Kunming mice was 17.4×10 7 ,See Figure 7 B. The results obtained by the two toxicity detection methods are similar. The toxicity of the botulinum toxin A protein prepared in this example is much higher than the minimum value of 1×10 7 U / mg, further indicating that the primary and higher structures of botulinum toxin A protein, including amino acid sequence, disulfide bond position, and spatial folding, are identical to those of the natural ones.

[0141] The above data show that the connecting peptide designed by the present invention can ensure the formation of correct disulfide bonds in botulinum toxin A, so that the recombinant botulinum toxin A protein has high biological activity. On the basis of the above examples, several other auxiliary sequences (polypeptide sequences between the first and second cleavage sites, also referred to as interenzyme polypeptides in this example) were also used, and corresponding expression clones were constructed and the target protein was prepared and activated. The effects of these auxiliary sequences and tag peptides on the biological activity of recombinant botulinum toxin were then analyzed (SEQ ID NO: 6 to SEQ ID NO: 12). The results showed that these flexible polypeptides were able to maintain the toxicity of the recombinant botulinum toxin at a very high level (12.7×10 7 ~17.6×10 7 U / mg), indicating that the use of these interenzyme peptides can obtain recombinant botulinum toxin protein A with high biological activity. Figure 7 C. Example 5 Preparation of recombinant botulinum toxin B protein

[0142] (1) Construction of recombinant botulinum toxin B protein and engineered bacteria

[0143] The protein structure of recombinant botulinum toxin B is similar to that of the recombinant botulinum toxin A protein in Example 1. Between the light and heavy chains, there is a first restriction enzyme cleavage site, a first auxiliary sequence, a tag peptide, a second auxiliary sequence, and a second restriction enzyme cleavage site. The connecting peptide used in this example to connect the light chain (SEQ ID NO: 13) and heavy chain (SEQ ID NO: 14) of botulinum toxin B has its N-terminus connected to the light chain and its C-terminus connected to the heavy chain. Its structure from N-terminus to C-terminus is as follows:

[0144] Kex2 protease cleavage site (KR)—first auxiliary sequence (SEQ ID NO: 3)—tag peptide (consisting of 8 histidines)—second auxiliary sequence (SEQ ID NO: 3)—Kex2 protease cleavage site (KR).

[0145] The recombinant botulinum toxin B polypeptide prepared in this example (its amino acid sequence is shown in SEQ ID NO: 15) has 16 Kex2 protease cleavage sites, 14 of which are also present in the natural protein sequence of botulinum toxin, such as Figure 8 shown.

[0146] The nucleotide coding sequence for the recombinant botulinum toxin B amino acid sequence was designed and optimized based on the characteristics of the host Escherichia coli, as shown in SEQ ID NO:16. This coding sequence was inserted into the pET-28 plasmid to generate the expression vector pET-28-RDS-B. After sequence verification, the expression vector was transformed into the host strain E. coli BL-21(DE3) (purchased from Thermo Fisher Scientific, USA).

[0147] (2) Protein expression and bacterial cell disruption

[0148] Recombinant botulinum toxin B engineered bacteria were cultured in shake flasks, and expression of recombinant botulinum toxin B was induced with 0.3 mM IPTG. The fermentation broth was centrifuged to obtain cells, which were then resuspended in 9-fold the cell weight of buffer (50 mM phosphate, 0.1 M NaCl, pH 7.0). The cells were disrupted using a high-pressure homogenizer (Shanghai Hongli Biotechnology Co., Ltd., 800-900 bar, twice) at low temperature. The lysate was centrifuged at 12,000 rpm for 40 minutes, and the supernatant was collected and filtered through a 0.45 μm filter to obtain the loading solution.

[0149] (3) First nickel affinity chromatography

[0150] The sample solution was passed through a pre-equilibrated nickel affinity chromatography column (Ni-IDAPurose 6Fast Flow Thousand Pure Biofiller), then washed with 3 column volumes of equilibration buffer (50mM phosphate, 0.1M NaCl, pH = 7.0), and then eluted with a step-by-step gradient. The eluent was prepared with equilibration buffer containing 50mM, 150mM, and 200mM imidazole (each gradient was 5 column volumes). The elution peaks of each section were collected separately, and the protein purity and content of each peak were detected by SDS-PAGE electrophoresis. The experimental results showed that the recombinant botulinum toxin B protein was mainly present in the eluent of 150mM imidazole, such as Figure 9 As shown in A.

[0151] (4) First anion chromatography

[0152] The eluate of 150mM imidazole was collected and diluted 3 times with 2mM Tris-HCl pH=7.0 buffer as an anion loading solution. The loading solution was passed through a pre-equilibrated anion chromatography column (Saifen 60Q), and then washed with an equilibration buffer (100mM Tris-HCl pH=7.0) until no protein solution flowed out. The elution buffer (100mM Tris-HCl 50mM NaCl pH=7.0) was used for 0-100% linear elution for 5CV, and then the elution buffer (100mM Tris-HCl 1M NaCl pH=7.0) was used for isocratic elution for 2-3 column volumes. The flow-through and eluate were collected, and the purity and content of the recombinant botulinum toxin B in each sample were detected by SDS-PAGE. The experimental results showed that the botulinum toxin B protein mainly appeared in the linear eluate, such as Figure 9 As shown in B.

[0153] (5) Removal of the connecting peptide

[0154] Collect the fractions containing relatively pure target protein in the anion chromatography, ultrafilter about 50 times with enzyme cleavage buffer (50mM Tris-HCl, pH=7.0), adjust the protein concentration to about 0.5mg / mL, and then add CaCl2 with a final concentration of 2mM. Add Kex2 protease and recombinant carboxypeptidase B (Shanghai Yaxin Biotechnology Co., Ltd.) at a ratio of 1:100 (enzyme: protein, w / w) and digest overnight at room temperature (25°C). The sample after enzyme digestion (nickel column loading solution) was subjected to reduction electrophoresis. The results showed that the protein after enzyme digestion was mainly 100KDa and 50KDa, while the protein molecular weight of the sample without protease was 150KDa, indicating that the protease cleaved the target protein. It also shows that the protein before enzyme cleavage was a single chain and non-toxic. Figure 10 As shown in A.

[0155] (6) Second nickel affinity chromatography

[0156] After the enzyme digestion is completed, the enzyme digestion solution is passed through a pre-equilibrated nickel affinity chromatography column (50mM Tris-HCl pH=7.0) once, the flow-through and washing solution (3 times the column volume) are collected, and EDTA (final concentration is 1mM) is added. Then, a step-by-step gradient elution is performed, and the eluents are respectively a solution containing 60mM NaCl and 50mM imidazole prepared with the equilibrium buffer (each gradient is 5 column volumes). The effluent peaks of each section are collected separately, and the enzyme digestion effect is detected by Western blot and SDS-PAGE. The SDS-PAGE results show that the protein after enzyme digestion is mainly in the effluent, and there are very few target protein bands in the eluate, indicating that most proteins do not carry a HIS tag. The Western blot test results also prove this point. Figure 10 shown.

[0157] (7) Second anion exchange column chromatography

[0158] The flow-through and washing liquid collected after the enzyme cleavage is completed and passed through the nickel affinity chromatography column are passed through a pre-equilibrated anion exchange column (Sefen 60Q, column equilibration liquid is 150mM Tris-HCl pH=7.0), and after loading, the equilibration buffer is used to rinse for 5 column volumes, and the flow-through peak is collected; then, the equilibration liquid is used to prepare 5 column volumes of 50mM NaCl for isocratic elution. The flow-through, washing liquid and eluate are collected, and the purity and content of the recombinant botulinum toxin B protein in each sample are detected by SDS-PAGE electrophoresis. The experimental results show that the eluate contains a lot of impurities, and the botulinum toxin B protein mainly appears in the flow-through and washing liquid, such as Figure 11 As shown in A. The relatively pure protein fractions in the collected flow-through and washing liquid were concentrated to about 5 mg / mL using a 50K ultrafiltration centrifuge tube (Sartorius), added with 10% glycerol and stored at -70°C until use.

[0159] (8) Superdex 200 gel filtration column chromatography

[0160] Under low temperature, the sample concentrated by the second anion exchange column chromatography was passed through a pre-equilibrated (50mM phosphate, 0.1M NaCl, pH=7.0) Superdex 200 column (Cytiva) and eluted with equilibration buffer at a flow rate of 0.5mL / min for 2 column volumes until no peak appeared. The elution peak appeared approximately 23 minutes later. Each elution peak was collected separately, and the protein purity and content of each peak were detected by SDS-PAGE electrophoresis. The experimental results showed that the botulinum toxin B protein was mainly concentrated in elution peak I, and elution peak II was mainly a 30KDa miscellaneous band, such as Figure 11 As shown in B. The reduction electrophoresis results of the final sample showed that the sample had two bands, which were the light chain and heavy chain of recombinant botulinum toxin B, that is, it reached electrophoretic purity, as shown in Figure 11 As shown in C. Example 6 Preparation of recombinant botulinum toxin E protein

[0161] (1) Construction of recombinant botulinum toxin E protein and engineered bacteria

[0162] The protein structure of recombinant botulinum toxin E is similar to that of the recombinant botulinum toxin A protein in Example 1. Between the light and heavy chains, there is a first restriction enzyme cleavage site, a first auxiliary sequence, a tag peptide, a second auxiliary sequence, and a second restriction enzyme cleavage site. The connecting peptide used in this example to connect the light chain (SEQ ID NO: 17) and heavy chain (SEQ ID NO: 18) of botulinum toxin E is connected to the light chain at its N-terminus and to the heavy chain at its C-terminus. Its structure from N-terminus to C-terminus is as follows:

[0163] Kex2 protease cleavage site (KR)—first auxiliary sequence (SEQ ID NO: 3)—tag peptide (consisting of 8 histidines)—second auxiliary sequence (SEQ ID NO: 3)—Kex2 protease cleavage site (KR).

[0164] The recombinant polypeptide prepared in this example (its amino acid sequence is shown in SEQ ID NO: 19) has 9 Kex2 protease cleavage sites, 7 of which are also present in the natural protein sequence of botulinum toxin, such as Figure 12 shown.

[0165] The nucleotide coding sequence for the recombinant botulinum toxin E amino acid sequence was designed and optimized based on the characteristics of the host Escherichia coli, as shown in SEQ ID NO:20. This coding sequence was inserted into the pET-28 plasmid to generate the expression vector pET-28-RDS-E. After sequence verification, the expression vector was transformed into the host strain E. coli BL-21(DE3) (purchased from Thermo Fisher Scientific, USA).

[0166] (2) Protein expression and bacterial cell disruption

[0167] Recombinant botulinum toxin E engineered bacteria were cultured in shake flasks, and expression of recombinant botulinum toxin E was induced with 0.3 mM IPTG. The fermentation broth was centrifuged to obtain cells, which were then resuspended in 9-fold the cell weight of buffer (50 mM phosphate, 0.1 M NaCl, pH 7.0). The cells were disrupted using a high-pressure homogenizer (Shanghai Hongli Biotechnology Co., Ltd., 800-900 bar, twice) at low temperature. The lysate was centrifuged at 12,000 rpm for 40 minutes, and the supernatant was collected and filtered through a 0.45 μm filter to obtain the loading solution.

[0168] (3) First nickel affinity chromatography

[0169] The sample solution was passed through a pre-equilibrated nickel affinity chromatography column (Biyuntian Biotechnology Co., Ltd.), then washed with 3 column volumes of equilibration buffer (50mM phosphate, 0.1M NaCl, pH = 7.0), and then eluted using a step-by-step gradient. The eluent was prepared with equilibration buffer containing 20mM, 50mM, and 200mM imidazole (each gradient was 5 column volumes). The elution peaks of each section were collected and the protein purity and content of each peak were detected by SDS-PAGE electrophoresis. The experimental results showed that the recombinant botulinum toxin E protein was mainly present in the eluents of 20mM and 50mM imidazole. Figure 13 shown.

[0170] (4) Removal of the connecting peptide

[0171] Fractions containing the target protein from the nickel affinity chromatography eluate were collected and ultrafiltered 100-fold using 50 mM phosphate buffer (pH 7.0). Kex2 protease and recombinant carboxypeptidase B were added at a 1:100 ratio (enzyme:protein, w / w) and digested at room temperature (25°C) for 5 h. The digested sample (nickel column load) was subjected to a second nickel affinity chromatography run.

[0172] (5) Second nickel affinity chromatography

[0173] After the digestion is complete, the digestion solution is passed through a pre-equilibrated nickel affinity chromatography column (column equilibration solution is 50mM phosphate buffer, pH=7.0) once, the flow-through and wash solution (3 times the column volume) are collected, and EDTA (final concentration is 1mM) is added. Each effluent peak is collected separately, and the digestion effect is detected by Western blot and SDS-PAGE. The results of SDS-PAGE reduction electrophoresis show that the protein after digestion is mainly in the effluent and wash solution, and the proteins are mainly 100KDa and 50KDa, indicating that the protease has cleaved the target protein. Most of the protein does not carry the HIS tag. Figure 14A. Western blot test results also proved this point, see Figure 14 As shown in B.

[0174] (6) Anion exchange column chromatography

[0175] The flow-through and washing liquid collected after the enzyme cleavage is completed and passed through the nickel affinity chromatography column are passed through the pre-equilibrated anion exchange column HQ (the column equilibration liquid is 50mM phosphate buffer, pH=7.0). After the sample is loaded, it is rinsed with the equilibration buffer until no protein flows out, and the flow-through peak is collected; then eluted with 1mol / L NaCl. The flow-through liquid, washing liquid and eluate are collected, and the purity and content of the recombinant botulinum toxin in each sample are detected by SDS-PAGE electrophoresis. The experimental results show that the eluate is mainly impurities, and the botulinum toxin E protein mainly appears in the flow-through, see Figure 15 A. The collected flow-through was concentrated to 5 mg / mL using a 50K ultrafiltration centrifuge tube (Sartorius), and 10% glycerol was added and stored at -70°C until use.

[0176] (7) Superdex 200 gel filtration column chromatography

[0177] The sample concentrated by anion exchange column chromatography was passed through a pre-equilibrated (50mM phosphate, 0.1M NaCl, pH=7.0) Superdex 200 column (Cytiva) and eluted with equilibration buffer at a flow rate of 0.5mL / min for 2 column volumes until no peak appeared. The elution peak appeared approximately 23 minutes later. Each elution peak was collected and the protein purity and content of each peak were detected by SDS-PAGE electrophoresis. The experimental results showed that the botulinum toxin E protein was mainly concentrated in elution peak I, and elution peak II was mainly a 50KDa miscellaneous band, as shown in Figure 2. Figure 15 B.

[0178] (8) Sample quantification and preservation

[0179] Peak I was collected from the molecular sieve chromatography elution, 10% glycerol was added, and the final protein concentration was determined to be 1-2 mg / mL using the A280 and BCA methods. The purity and residual HIS tag of the final product were tested by SDS-PAGE and Western blotting. The results showed that the sample showed a single band in the non-reduced electrophoresis, indicating that it was electrophoretically pure. The reduced electrophoresis showed only 100 kDa (heavy chain) and 50 kDa (light chain) bands. Figure 16 A. WB results showed that the HIS tag residues were less than 1‰, see Figure 16 B. Samples were aliquoted and stored in a -70°C freezer. The sequence information involved in this article is as follows: SEQ ID NO: 1 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITS SEQ ID NO:2 ALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL SEQ ID NO:3 GTEEAEKLG SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 HHHHHHHH SEQ ID NO:7 GGSHHHHHHHHGGS SEQ ID NO:8 GTEEAEKLGHHHHHHHH SEQ ID NO:9 HHHHHHHHGTEEAEKLG SEQ ID NO:10 GSGSEEGSGEGTEEAEKLGHHHHHHHGSGSEEGSGEGTEEAEKLG SEQ ID NO:11 DDADHHHHHHHHDDAD SEQ ID NO:12 TEDAEKLHHHHHHHHTEDAEKL SEQ ID NO:13 MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLE MIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGY FSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNINIYKNKFKDKYKF VEDSEGKYSIDVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQAYEEISKEHLAVYQMCKSV SEQ ID NO:14APGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVNDFVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLIPVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE SEQ ID NO:15 SEQ ID NO:16 SEQ ID NO:17 MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHRFGSIAIVTFSPEYSFRFNDNCMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIV SEQ ID NO:18 SICIEINGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADISIVVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKIINEVKINKLREYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILI SYFNKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNISQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEIIWTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNLGNIHVSDNILFKIVNCSSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYLLYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDNLVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNCTMNFKNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK SEQ ID NO:19 SEQ ID NO:20

Claims

1. A recombinant polypeptide comprising a light chain of a neurotoxin and / or a heavy chain of a neurotoxin, and a connecting peptide; The connecting peptide comprises a tag peptide segment and at least one or two enzyme cleavage site sequences selected from the following: a first enzyme cleavage site sequence and a second enzyme cleavage site sequence; After the first cleavage site sequence is recognized by the protease, the protease cuts at the connection between the light chain and the connecting peptide, and / or after the second cleavage site sequence is recognized by the protease, the protease cuts at the connection between the connecting peptide and the heavy chain.

2. The recombinant polypeptide according to claim 1, which comprises a light chain and a heavy chain of a neurotoxin and a connecting peptide located between the light chain and the heavy chain, for example, from the N-terminus to the C-terminus, comprises a light chain of a neurotoxin, a connecting peptide and a heavy chain of a neurotoxin, so that the light chain and the heavy chain are connected into a polypeptide chain by the connecting peptide.

3. The recombinant polypeptide according to claim 1 or 2, comprising the light chain of the neurotoxin connected to a connecting peptide, for example comprising the light chain of the neurotoxin and the connecting peptide from the N-terminus to the C-terminus, and / or It comprises the heavy chain of the neurotoxin connected to a connecting peptide, for example comprising the connecting peptide and the heavy chain of the neurotoxin from the N-terminus to the C-terminus, The connecting peptide connected to the light chain and the connecting peptide connected to the heavy chain are the same or different. The recombinant polypeptide of claim 1 , wherein the light chain and the heavy chain are located in separate polypeptide chains.

5. The recombinant polypeptide according to any one of claims 1 to 4, wherein the neurotoxin is selected from the group consisting of botulinum neurotoxin, tetani neurotoxin and diphtheria toxin.

6. The recombinant polypeptide according to claim 5, wherein the botulinum neurotoxin is selected from serotypes A, B, C, D, E, F and G, preferably from serotypes A, B and E, such as serotype A botulinum neurotoxin.

7. The recombinant polypeptide according to any one of claims 1 to 6, wherein the amino acid sequence of the light chain is the amino acid sequence shown in SEQ ID NO: 1, and / or the amino acid sequence of the heavy chain is the amino acid sequence shown in SEQ ID NO:

2.

8. The recombinant polypeptide according to any one of claims 1 to 7, wherein the first restriction enzyme cleavage site sequence and the second restriction enzyme cleavage site sequence are independently selected from the restriction enzyme cleavage site sequences of Kex2 enzyme, EK enzyme, and TEV enzyme, For example, the first restriction enzyme cleavage site sequence is selected from R, K, RR, KK, RK and KR, and / or the second restriction enzyme cleavage site is selected from R, K, RR, KK, RK, KR and DDDDK. 9 . The recombinant polypeptide according to claim 8 , wherein the first restriction enzyme cleavage site sequence and the second restriction enzyme cleavage site sequence are both restriction enzyme cleavage site sequences of Kex2 enzyme.

10. The recombinant polypeptide according to any one of claims 1 to 9, wherein the tag peptide segment is selected from a polyhistidine tag (poly(His)-tag), a FLAG tag (FLAG-tag), a streptavidin binding tag (Strep-tag), and a glutathione S-transferase tag (GST-tag). The recombinant polypeptide according to claim 10 , wherein the tag peptide is selected from a polyhistidine tag (poly(His)-tag), such as 4, 5, 6, 7, 8, 9 or 10 polyhistidine. 12 . The recombinant polypeptide according to claim 1 , further comprising a first auxiliary sequence, wherein the first linker sequence is located between the first restriction enzyme cleavage site sequence and the tag peptide segment.

13. The recombinant polypeptide according to any one of claims 1 to 12, further comprising a second auxiliary sequence, wherein the second auxiliary sequence is located between the second restriction enzyme cleavage site sequence and the tag peptide segment.

14. The recombinant polypeptide of claim 12 or 13, wherein the first and second helper sequences are each independently at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length.

15. The recombinant polypeptide according to any one of claims 12 to 14, wherein the first auxiliary sequence and the second auxiliary sequence are each independently selected from GSGS, EEGSGS, GSGSDDGSGS, GSGSEDGSGS, GSGSDEGSGS, EEAE, DDAD, TEEAEKL, TDDADKL, TEDAEKL, TDEADKL, GTEEAEKLG, EGTEEAEKLG, EGTDDADKLG, EGTEEADKLG, EGTDDAEKLG or EGTEDADKLG and EGTDEAEKLG, and sequences having at least 60%, 70%, 80%, or 90% identity to any of the above sequences.

16. An isolated polynucleotide encoding the recombinant polypeptide according to any one of claims 1 to 15. An expression vector comprising the polynucleotide according to claim 16 .

18. A cell comprising the polynucleotide according to claim 16 or the expression vector according to claim 17.

19. The cell according to claim 18, wherein the cell is selected from prokaryotic cells and eukaryotic cells; for example, the prokaryotic cell is selected from Escherichia coli cells; for example, the eukaryotic cell is selected from yeast cells, insect cells, plant cells, mammalian cells.

20. A method for producing a neurotoxin, comprising culturing the cell according to claim 18 or 19 under conditions suitable for expression of the recombinant polypeptide.

21. The method according to claim 20, further comprising isolating the recombinantly expressed recombinant polypeptide and cleaving the recombinant polypeptide with a protease.

22. The method of claim 20 or 21, further comprising purifying the neurotoxin from the cell culture.

23. A pharmaceutical or cosmetic composition comprising a neurotoxin produced according to any one of claims 20 to 22.

24. Use of the recombinant polypeptide of any one of claims 1 to 15, the polynucleotide of claim 16, the expression vector of claim 17, or the cell of claim 18 or 19 in the preparation of a neurotoxin, for example, the neurotoxin is selected from tetanus neurotoxin, diphtheria toxin and botulinum neurotoxin.

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