Recombinant polypeptide and method for preparing neurotoxin

Abstract

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

Description

A recombinant polypeptide and a method for preparing neurotoxin

[0001] Cross-references to related applications

[0002] 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

[0003] 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

[0004] 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.

[0005] 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.

[0006] 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 zinc metalloprotease with a relative molecular mass of 50 kDa and is the active component of BoNT. The HC, with a relative molecular mass 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 subunit, respectively.

[0007] 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.

[0008] 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).

[0009] 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.

[0010] 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.

[0011] Summary of the Invention

[0012] 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.

[0013] 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.

[0014] 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;

[0015] 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;

[0016] 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.

[0017] 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.

[0018] 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

[0019] 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,

[0020] The connecting peptide connected to the light chain and the connecting peptide connected to the heavy chain are the same or different.

[0021] In a specific embodiment, said light chain and said heavy chain are in separate polypeptide chains.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] In a second aspect, the present invention provides an isolated polynucleotide encoding the recombinant polypeptide according to the first aspect of the present invention.

[0032] 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.

[0033] In a third aspect, the present invention provides an expression vector comprising the polynucleotide according to the second aspect of the present invention.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] In one embodiment, the method further comprises isolating the recombinantly expressed recombinant polypeptide and cleaving the recombinant polypeptide with a protease.

[0038] In another embodiment, the method further comprises purifying the neurotoxin from the cell culture.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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

[0043] Figure 1 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 a schematic diagram of 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.

[0044] Figure 2 shows the results of nickel affinity chromatography purification of recombinant botulinum toxin A and analysis of its stability to enzyme cleavage; wherein, A is an electrophoretic diagram of the nickel affinity chromatography purification results, with the arrow indicating recombinant botulinum toxin; B is an electrophoretic diagram of the Kex2 protease-cleaved purified sample; C is a Western blot diagram of the enzyme cleavage results; the sample containing the HIS tag before enzyme cleavage (0 min) was loaded at 100 ng, and the sample loading amount for other samples was 3 μg. In each figure, M represents the protein molecular weight marker.

[0045] Figure 3 shows the results of recombinant botulinum toxin A after enzymatic cleavage and a second nickel column affinity chromatography purification; wherein, A is a reduced SDS-PAGE electrophoresis analysis after enzymatic cleavage, B is a Western blot analysis of the enzymatic cleavage effect (the sample loading amount of the protein with the HIS tag before enzymatic cleavage was 30 ng, and the sample loading amount of other samples was 3 μg), C is a reduced SDS-PAGE electrophoresis of nickel column affinity chromatography after enzymatic cleavage; D is an electrophoresis of the recombinant protein with the natural botulinum toxin sequence 438-448 (treated at room temperature for 3 hours and then run on SDS-PAGE electrophoresis). In each figure, M is the protein molecular weight marker.

[0046] Figure 4 shows the results of anion HQ column chromatography purification; wherein 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.

[0047] Figure 5 shows the purity and HIS residue detection results of the prepared recombinant botulinum toxin A; wherein 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 a protein molecular weight marker.

[0048] Figure 6 shows the primary structure detection results of recombinant botulinum toxin A prepared by mass spectrometry analysis; wherein A is the protein's precise molecular weight detection result, B is the C430 / 454 disulfide bond position detection result, C is the C1235 / 1280 disulfide bond position detection result, and D is the protein's N- and C-terminal amino acid sequence detection result.

[0049] Figure 7 shows the toxicity test results of the recombinant botulinum toxin A produced by 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; and C is the toxicity test results of the recombinant botulinum toxin A protein activated with various auxiliary sequences.

[0050] FIG8 shows the Kex2 protease cleavage site in the recombinant botulinum toxin B protein for preparing recombinant botulinum toxin B according to the present invention.

[0051] Figure 9 shows the results of the first nickel affinity chromatography and anion exchange purification of recombinant botulinum toxin B. A is the electrophoresis diagram of the first nickel affinity chromatography; B is the electrophoresis diagram of the first anion HQ column chromatography.

[0052] Figure 10 shows the results of enzymatic digestion and secondary nickel-affinity chromatography purification of recombinant botulinum toxin B. Figure A shows an SDS-PAGE electrophoresis of samples digested with Kex2 protease and purified by secondary nickel-affinity chromatography; Figure 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.

[0053] Figure 11 shows the results of the second purification of recombinant botulinum toxin B by anion column and molecular sieve chromatography. A is the electropherogram of the separation and purification results using an HQ column; B is the electropherogram of the separation and purification results using a Superdex 200 column; and C is the reduced SDA-PAGE electropherogram of the prepared recombinant botulinum toxin B. In each figure, M is a protein molecular weight marker.

[0054] FIG12 shows the Kex2 cleavage site in the recombinant botulinum toxin E protein for preparing recombinant botulinum toxin E according to the present invention.

[0055] Figure 13 shows the results of nickel affinity chromatography purification of recombinant botulinum toxin E. M represents the protein molecular weight marker.

[0056] Figure 14 shows the 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 of the HIS-tagged protein is 30 ng, and the sample loading amount of other samples is 3 μg), and M is the protein molecular weight marker.

[0057] Figure 15 shows the results of purification of recombinant botulinum toxin E by anion column and molecular sieve chromatography; wherein 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.

[0058] Figure 16 shows the purity and HIS residue detection results of the prepared recombinant botulinum toxin E; wherein A is an SDA-PAGE electrophoresis diagram, and B is a Western blot analysis diagram (the "positive" sample is a purified, non-enzymatically digested, HIS-tagged recombinant botulinum toxin protein). In each figure, M is the protein molecular weight marker. DETAILED DESCRIPTION

[0059] definition

[0060] 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, and 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] As an example, this application involves the following technical solutions:

[0071] 1. A polypeptide for recombinant expression to produce neurotoxin protein, characterized in that

[0072] Tag peptides, which are used for affinity purification;

[0073] 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;

[0074] The second enzyme cleavage site includes at least one amino acid and is located at the C-terminal side of the tag peptide segment.

[0075] 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.

[0076] 3. The polypeptide according to embodiment 2, characterized in that m and n are each selected from natural numbers 1 to 10.

[0077] 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.

[0078] 5. The polypeptide according to embodiment 2, characterized in that the tag peptide is selected from HHHHHH, HHHHHHH or HHHHHHHH.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 10. The polypeptide according to embodiment 1, characterized in that the first restriction enzyme cleavage site is a Kex2 restriction enzyme cleavage site.

[0084] 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.

[0085] 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.

[0086] 13. The polypeptide according to embodiment 1, characterized in that the flexible sequence comprises at least one glycine or serine.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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%.

[0092] 19. The polypeptide according to embodiment 1, further comprising:

[0093] A first auxiliary sequence, which is located between the tag peptide segment and the first enzyme cleavage site; and

[0094] The second auxiliary sequence is located between the tag peptide segment and the second enzyme cleavage site.

[0095] 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%.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 25. The polypeptide according to embodiment 1, characterized in that the prokaryotic organism is Escherichia coli.

[0101] 26. The polypeptide according to embodiment 25, characterized in that the Escherichia coli is selected from BL21(DE3), HMS174(DE3), pLysS or pLysE.

[0102] 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

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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%.

[0110] 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;

[0111] The artificial polypeptide includes the polypeptide described in embodiment 1.

[0112] 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.

[0113] 36. The method for producing neurotoxin protein by recombinant expression according to embodiment 34 is characterized in that the expression system is Escherichia coli.

[0114] 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.

[0115] Example

[0116] 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.

[0117] Example 1 Construction of recombinant botulinum toxin A protein and engineered bacteria

[0118] Figure 1A 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:

[0119] 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).

[0120] The recombinant polypeptide prepared in this example (whose amino acid sequence is shown in SEQ ID NO: 4) has 9 Kex2 protease cleavage sites, 7 of which are also present in the native protein sequence of botulinum toxin, as shown in FIG1B .

[0121] The nucleotide coding sequence for 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. This coding sequence was inserted into the pET-28 plasmid to generate the expression vector pET-28-RDS, as shown in Figure 1C. After sequence verification, the expression vector was transformed into the host strain E. coli BL-21 (DE3) (purchased from Thermo Fisher Scientific, USA).

[0122] Example 2 Preparation of active recombinant botulinum toxin A protein

[0123] 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:

[0124] (1) Bacteria disruption

[0125] 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.

[0126] (2) Nickel affinity chromatography

[0127] In a chromatography cabinet at 4°C, the sample was passed through a pre-equilibrated nickel affinity chromatography column (Biyuntian Biotechnology Co., Ltd.). The column was then washed with 5 column volumes of equilibration buffer (50 mM phosphate, 0.1 M NaCl, pH 7.0) until no protein remained. Elution was then performed using a stepwise gradient with equilibration buffer containing 20, 50, 200, and 500 mM imidazole (5 column volumes per step). The peaks were collected and analyzed for protein purity and content by SDS-PAGE electrophoresis. The results showed that the recombinant botulinum toxin protein was primarily present in the 50 mM imidazole eluate, demonstrating high purity (see Figure 2A). The 50 mM imidazole eluate was collected and ultrafiltered 100 times against 50 mM phosphate buffer (pH 7.0). Kex2 protease was added at a ratio of 1:500 (enzyme:protein, w / w) and incubated for 5 hours. Samples were taken periodically for electrophoresis to detect the nonspecific cleavage of the target protein by the protease. The inventors unexpectedly discovered that after 5 hours of cleavage, the amount of target protein remained largely unchanged, indicating that the nonspecific cleavage effect of Kex2 on the target protein was very weak. Almost no significant cleavage of the recombinant polypeptide at the Kex2 cleavage site present in native botulinum toxin was observed (see Figure 2B). Western blot analysis revealed that the HIS tag removal of the target protein gradually decreased with increasing incubation time. After 3 hours of cleavage (3 μg sample loading), the rate of reduction significantly slowed, and the band intensity at this point was weaker than that of the sample before cleavage (100 ng sample loading). This indicates that after 3 hours of incubation with the protease, over 96% of the target protein had been cleaved of the HIS tag (see Figure 2C).

[0128] It can be seen that this example has verified that the scheme of using Kex2 protease to remove exogenous polypeptides is feasible.

[0129] (3) Removal of the connecting peptide

[0130] 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). The enzyme cleavage buffer was added to make the botulinum toxin protein concentration at 0.5mg / ml, and the enzyme cleavage was carried out at 25°C for 3 hours. Reduction electrophoresis of the digested sample (nickel column load) revealed that the cleaved proteins were primarily 100 kDa and 50 kDa, while the protein in the sample without protease had a molecular weight of 150 kDa, indicating that the protease cleaved the target protein and that the protein before cleavage was a single chain and non-toxic (see Figure 3A). Western blot analysis using a His tag antibody further examined the effect of HIS tag removal. The results showed that although the sample load (3 μg) after 3 hours of digestion was 100 times that of the positive protein (30 ng), the target band intensity in the former was significantly lower than that in the latter, indicating that over 99% of the HIS tag was removed (see Figure 3B).

[0131] After the enzymatic digestion was completed, the digestion solution was passed once through a pre-equilibrated nickel affinity chromatography column (column equilibration solution was 50 mM phosphate buffer, pH = 7.0) at low temperature. The flow-through and wash solution (3 column volumes) were collected, and EDTA (final concentration was 1 mM) was added. The enzymatic digestion effect was detected by Western blot and SDS-PAGE. The SDS-PAGE results showed that the protein after enzymatic digestion was mainly in the effluent, and there were very 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, see Figures 3B and 3C.

[0132] The recombinant protein of this example lacks the amino acid sequence 439-448 of native botulinum toxin A. Our experimental results show that when expressed in E. coli, recombinant botulinum toxin A protein containing this native sequence is cleaved, forming two chains and exhibiting toxin activity, even without the addition of proteases (see Figure 3D). This leads to the premature appearance of toxic forms during recombinant botulinum toxin A expression, significantly increasing the biosafety risks of the production process.

[0133] (4) Anion exchange column chromatography

[0134] At low temperature, the flowthrough and washes collected after enzymatic digestion and nickel affinity chromatography were passed through a pre-equilibrated anion exchange column, HQ (equilibrated with 50 mM phosphate buffer, pH 7.0). After sample loading, the column was rinsed with equilibration buffer for 5-10 column volumes, and the flowthrough peak was collected. Elution was then performed using a linear gradient of 0 to 1 mol / L NaCl over a total of 20 column volumes, with the elution peak appearing approximately 5 column volumes later. The flowthrough, washes, and eluates were collected and analyzed by SDS-PAGE electrophoresis to determine the purity and content of the recombinant botulinum toxin protein in each sample. The results showed that the eluate was primarily composed of impurities, while the botulinum toxin protein was primarily present in the flowthrough, with a purity of approximately 95% (see Figure 4A). The collected flowthrough was concentrated using 50K ultrafiltration centrifuge tubes (Sartorius) to a protein concentration of 5 mg / ml, added with 10% glycerol, and stored at -70°C until use.

[0135] (5) Superdex 200 gel filtration column chromatography

[0136] At low temperature, the concentrated sample was passed through a pre-equilibrated Superdex 200 column (Cytiva) (50 mM phosphate, 0.1 M NaCl, pH 7.0). Elution was performed with equilibration buffer at a flow rate of 0.5 ml / min for two column volumes until no peak appeared. The elution peak appeared approximately 23 minutes later. Each elution peak was collected and analyzed by SDS-PAGE electrophoresis for protein purity and content. The results showed that the botulinum toxin protein was primarily concentrated in elution peak I, with virtually no contaminants. Elution peaks II and III were primarily composed of 55 kDa and 30 kDa contaminants, respectively, as shown in Figure 4B.

[0137] (6) Sample quantification and preservation

[0138] Peak I, eluted from molecular sieve chromatography, was collected and 10% glycerol was added. The final protein concentration was quantified using the A280 and BCA assays, with a final protein concentration of 1-2 mg / mL. The purity and residual HIS tag of the final product were assessed by SDS-PAGE, HPLC, and Western blotting. The results indicated that the samples were electrophoretically pure, with a single band observed. Western blotting revealed less than 1‰ of the HIS tag residue, and HPLC analysis revealed a purity of 99.19% (see Figure 5). The samples were stored in aliquots at -70°C.

[0139] 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.

[0140] Example 3 Sequence Identification of Recombinant Botulinum Toxin A Protein

[0141] Botulinum toxin A protein consists of two polypeptide chains connected by a pair of interchain disulfide bonds. Samples were treated with or without the addition of the reducing agent TCEP (TCEP can break the interchain disulfide bonds, separating the two chains and placing the sample in a reduced state). Protein samples were centrifuged at 12,000 g for 30 minutes and then separated using an ACQUITY UPLC Protein BEH 300C4 column and transferred to a Xevo G2-XS Q-TOF (Waters) mass spectrometer to determine the intact and reduced molecular weights of the botulinum toxin A protein prepared in this example. Mass spectrometric data were analyzed after deconvolution using UNIFI software; the specific data are shown in Figure 6A. 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.

[0142] 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 the necessary biological activity. Nanoliter liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) was used to identify the intrachain and interchain disulfide bond pairing modes of the botulinum toxin A protein prepared in this embodiment. The nanoliter liquid chromatography and mass spectrometer were EASY-nLC 1200 and Thermo Orbitrap Q Exactive HF from Thermo Corporation of the United States, respectively, and the liquid phase analysis column was a C18 reverse phase column. The mass spectrometry data were analyzed by BioPharma Finder (V5.2) software, and rich primary and secondary ion information was collected for each pair of disulfide bonds. The primary mass deviation of the ion signal value was 10ppm, and the secondary mass deviation was 20ppm. The results ( FIG6B ) show that the botulinum toxin A prepared in this example forms a disulfide bond between cysteine ​​429 on the light chain and cysteine ​​6 on the heavy chain (corresponding to the disulfide bond formed between cysteine ​​430 and 454 in the native botulinum toxin protein), and also between cysteine ​​787 and cysteine ​​832 on the heavy chain (corresponding to the disulfide bond formed between cysteine ​​1235 and 1280 in the native botulinum toxin A protein). For detailed values, see FIG6C . These results demonstrate that the botulinum toxin protein prepared in this example contains one pair of interchain disulfide bonds and one pair of intrachain disulfide bonds, and that the disulfide bond linkages are consistent with theoretical results.

[0143] The amino acid sequence is the basis for the function of the protein. The results in Figure 6-A 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, reduced, and hydrolyzed by protease Glu-C and Trypsin with guanidine hydrochloride, 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 for protein N-terminal and C-terminal amino acid sequence determination, and the mass spectrometry data was 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 are consistent with the theoretical sequence, as shown in Figure 6D. This also shows that after enzyme cleavage, all exogenous amino acid sequences are removed.

[0144] Example 4 Toxicity Determination of Recombinant Botulinum Toxin A Protein

[0145] 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 7A.

[0146] Intraperitoneal injection:

[0147] 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:

[0148] 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 7B. 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.

[0149] 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 inter-enzyme peptides can obtain recombinant botulinum toxin protein A with high biological activity, as shown in Figure 7C.

[0150] Example 5 Preparation of recombinant botulinum toxin B protein

[0151] (1) Construction of recombinant botulinum toxin B protein and engineered bacteria

[0152] 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:

[0153] 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).

[0154] The recombinant botulinum toxin B polypeptide prepared in this example (whose amino acid sequence is shown in SEQ ID NO: 15) has 16 Kex2 protease cleavage sites, 14 of which are also present in the native protein sequence of botulinum toxin, as shown in FIG8 .

[0155] 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).

[0156] (2) Protein expression and bacterial cell disruption

[0157] 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.

[0158] (3) First nickel affinity chromatography

[0159] The sample was passed through a pre-equilibrated nickel affinity chromatography column (Ni-IDA Purose 6 Fast Flow BioPure Packing), then washed with 3 column volumes of equilibration buffer (50mM phosphate, 0.1M NaCl, pH 7.0). Elution was then performed using a stepwise gradient. The eluents were prepared using equilibration buffer containing 50mM, 150mM, and 200mM imidazole (5 column volumes for each gradient). The peaks were collected from each step and the protein purity and content of each peak were determined by SDS-PAGE electrophoresis. The results showed that the recombinant botulinum toxin B protein was primarily present in the 150mM imidazole eluate, as shown in Figure 9A.

[0160] (4) First anion chromatography

[0161] The 150mM imidazole eluate was collected and diluted three-fold with 2mM Tris-HCl (pH 7.0) as the anion loading buffer. The load was passed through a pre-equilibrated anion chromatography column (Serifen 60Q), then washed with equilibration buffer (100mM Tris-HCl (pH 7.0) until protein-free. Elution was then performed using elution buffer (100mM Tris-HCl, 50mM NaCl (pH 7.0)) with a 0-100% linear elution rate for 5CV, followed by isocratic elution using elution buffer (100mM Tris-HCl, 1M NaCl (pH 7.0)) for 2-3 column volumes. The flow-through and eluate were collected, and the purity and content of recombinant botulinum toxin B in each sample were determined by SDS-PAGE. The results showed that botulinum toxin B protein was primarily present in the linear elution, as shown in Figure 9B.

[0162] (5) Removal of the connecting peptide

[0163] The fraction containing the purer target protein from the anion chromatography was collected and ultrafiltered about 50 times with enzyme digestion buffer (50mM Tris-HCl, pH=7.0) to adjust the protein concentration to about 0.5mg / mL. Then, CaCl2 was added to it at a final concentration of 2mM. Kex2 protease and recombinant carboxypeptidase B (Shanghai Yaxin Biotechnology Co., Ltd.) were added at a ratio of 1:100 (enzyme:protein, w / w) and digested overnight at room temperature (25°C). The sample after digestion (nickel column loading solution) was subjected to reduction electrophoresis. The results showed that the protein after digestion was mainly at 100KDa and 50KDa, while the protein molecular weight of the sample without protease was 150KDa, indicating that the protease cleaved the target protein. At the same time, it was shown that the protein before digestion was a single chain and non-toxic, as shown in Figure 10A.

[0164] (6) Second nickel affinity chromatography

[0165] After the digestion is complete, the digestion solution is passed through a pre-equilibrated nickel affinity chromatography column (50mM Tris-HCl pH = 7.0) once, the flow-through and wash solution (3 column volumes) are collected, and EDTA (final concentration of 1mM) is added. Then, a step-by-step gradient elution is performed, with the eluents being 60mM NaCl and 50mM imidazole solutions prepared with the equilibration buffer (each gradient is 5 column volumes). The effluent peaks of each section are collected, and the digestion effect is detected by Western blot and SDS-PAGE. The SDS-PAGE results show that the protein after 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 confirm this, as shown in Figure 10.

[0166] (7) Second anion exchange column chromatography

[0167] After the enzyme digestion and nickel affinity chromatography column, the collected flowthrough and washes were passed through a pre-equilibrated anion exchange column (Separate 60Q, equilibrated with 150 mM Tris-HCl, pH 7.0). After sample loading, the column was rinsed with equilibration buffer for 5 column volumes, and the flowthrough peak was collected. Elution was then performed isocratically for 5 column volumes using equilibration buffer containing 50 mM NaCl. The flowthrough, washes, and eluates were collected and analyzed by SDS-PAGE electrophoresis to determine the purity and content of the recombinant botulinum toxin B protein in each sample. The results showed that the eluate contained a high concentration of contaminants, with botulinum toxin B protein primarily present in the flowthrough and washes, as shown in Figure 11A. The purer protein fractions from the collected flowthrough and washes were concentrated to approximately 5 mg / mL using 50K ultrafiltration centrifuge tubes (Sartorius), added with 10% glycerol, and stored at -70°C until use.

[0168] (8) Superdex 200 gel filtration column chromatography

[0169] At low temperature, the sample, concentrated after the second anion exchange column chromatography, was passed through a pre-equilibrated (50 mM phosphate, 0.1 M NaCl, pH = 7.0) Superdex 200 column (Cytiva). Elution was performed with equilibration buffer at a flow rate of 0.5 mL / min for two 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 B protein was primarily concentrated in elution peak I, while elution peak II was primarily a 30 kDa miscellaneous band, as shown in Figure 11B. The final sample was subjected to reduced electrophoresis, indicating that the sample was electrophoretically pure, with two bands representing the light and heavy chains of recombinant botulinum toxin B, as shown in Figure 11C.

[0170] Example 6 Preparation of recombinant botulinum toxin E protein

[0171] (1) Construction of recombinant botulinum toxin E protein and engineered bacteria

[0172] 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:

[0173] 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).

[0174] The recombinant polypeptide prepared in this example (whose 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, as shown in FIG12 .

[0175] 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).

[0176] (2) Protein expression and bacterial cell disruption

[0177] 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.

[0178] (3) First nickel affinity chromatography

[0179] The sample was passed through a pre-equilibrated nickel affinity chromatography column (Biyuntian Biotechnology Co., Ltd.), then washed with three column volumes of equilibration buffer (50 mM phosphate, 0.1 M NaCl, pH 7.0). Elution was then performed using a stepwise gradient with eluents prepared in equilibration buffer containing 20 mM, 50 mM, and 200 mM imidazole (5 column volumes for each gradient). The peaks were collected from each step and the protein purity and content of each peak were determined by SDS-PAGE electrophoresis. The results showed that recombinant botulinum toxin E protein was primarily present in the eluents containing 20 mM and 50 mM imidazole, as shown in Figure 13.

[0180] (4) Removal of the connecting peptide

[0181] 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.

[0182] (5) Second nickel affinity chromatography

[0183] After the digestion was complete, the digestion solution was passed through a pre-equilibrated nickel affinity chromatography column (column equilibration solution was 50mM phosphate buffer, pH = 7.0) once, the flow-through and wash solution (3 column volumes) were collected, and EDTA (final concentration was 1mM) was added. The effluent peaks of each section were collected separately, and the digestion effect was detected by Western blot and SDS-PAGE. The SDS-PAGE reduction electrophoresis results showed that the proteins after digestion were mainly in the effluent and wash solution, and the proteins were mainly at 100KDa and 50KDa, indicating that the protease cleaved the target protein and most of the protein did not carry the HIS tag, as shown in Figure 14A. The Western blot detection results also confirmed this, as shown in Figure 14B.

[0184] (6) Anion exchange column chromatography

[0185] 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), and after loading, it is rinsed with the equilibration buffer until no protein liquid 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 liquid, as shown in Figure 15A. The collected flow-through liquid is concentrated to 5mg / mL using a 50K ultrafiltration centrifuge tube (Sartorius), and 10% glycerol is added and stored at -70°C for use.

[0186] (7) Superdex 200 gel filtration column chromatography

[0187] The sample, concentrated by anion exchange chromatography, was passed through a Superdex 200 column (Cytiva) pre-equilibrated (50 mM phosphate, 0.1 M NaCl, pH 7.0). Elution was performed with equilibration buffer at a flow rate of 0.5 mL / min for two column volumes until no peak appeared. The elution peak appeared approximately 23 minutes later. Each elution peak was collected and analyzed by SDS-PAGE electrophoresis for protein purity and content. The results showed that the botulinum toxin E protein was primarily concentrated in elution peak I, while elution peak II was primarily a 50 kDa contaminant (see Figure 15B).

[0188] (8) Sample quantification and preservation

[0189] Collect the molecular sieve chromatography elution peak I, add 10% glycerol, and quantify it using the A280 and BCA methods to determine the final protein concentration at 1-2 mg / mL. The purity of the final product and the residual HIS tag were detected by SDS-PAGE and WB methods. The results showed that the non-reduced electrophoresis of the sample showed a single band, that is, it was electrophoretically pure, and the reduced electrophoresis only had 100KDa (heavy chain) and 50KDa (light chain) bands, as shown in Figure 16A. The WB results showed that the HIS tag residues were less than 1‰, as shown in Figure 16B. The samples were stored in a -70°C refrigerator.

[0190] The sequence information involved in this article is as follows:

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 restriction enzyme cleavage site sequences selected from the following: a first restriction enzyme cleavage site sequence and a second restriction 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 one 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 according to 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.

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 site sequence and the second restriction site sequence are independently selected from the restriction 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).

11. The recombinant polypeptide according to claim 10, wherein the tag peptide segment 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 any one of claims 1 to 11, further comprising a first auxiliary sequence, wherein the first linker sequence is located between the first restriction 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 site sequence and the tag peptide segment.

14. The recombinant polypeptide of claim 12 or 13, wherein the first auxiliary sequence and the second auxiliary sequence 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 the method of 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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