Recombinant botulinum toxin and preparation method thereof

Abstract

The invention relates to the technical field of a preparation method of a type A recombinant botulinum toxin, in particular to a type A botulinum toxin obtained by adopting a gene recombination technology, and the amino acid sequence of the type A botulinum toxin is consistent with the sequence of the type A botulinum toxin prepared by extracting and purifying a wild strain Clostridium botulinum. According to the technical scheme, the single-chain protein of the botulinum toxin A sequentially comprises a botulinum toxin A light chain or a modified botulinum toxin A light chain, a connecting region and a botulinum toxin A heavy chain. According to the scheme, the high-quality recombinant botulinum toxin A is obtained after the single-chain protein is subjected to fermentation, crude purification, enzyme digestion and fine purification. According to the technical scheme, the problems of complex process, long period, high safety risk and the like in the existing process for preparing the botulinum toxin A from the wild bacterial strain clostridium botulinum can be solved, and meanwhile, the problem that the amino acid sequence consistent with that of the botulinum toxin A prepared from the wild bacterial strain clostridium botulinum cannot be obtained through the existing gene recombination technology is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of methods for preparing recombinant botulinum toxin, and in particular to a type A botulinum toxin obtained by genetic recombination technology, the amino acid sequence of which is consistent with the amino acid sequence of the type A botulinum toxin prepared by extraction and purification of wild strains of Clostridium botulinum. Background Art

[0002] Natural botulinum toxin type A (BoNT / A) is a strong neurotoxin produced by wild-type Clostridium botulinum and is commonly used in the medical field. Botulinum toxin type A is mainly used to improve facial spasms, cerebral palsy, blepharospasm, cervical dystonia, etc. by blocking neurotransmitter transmission and inhibiting muscle contraction. It is also used to treat certain types of headaches, hyperhidrosis and some urinary system diseases.

[0003] In the prior art, there are mainly two preparation methods:

[0004] One is to obtain it through the culture and extraction of wild-type Clostridium botulinum, which is the main method for the current marketed botulinum toxin type A products, such as Botox, Xeomin, Dysport, etc. When using Clostridium botulinum to prepare botulinum toxin type A, the laboratory and preparation process are very demanding due to safety issues, and the preparation process of this method is long and complicated; wild-type Clostridium botulinum also produces hemagglutinin (HA) and non-toxic non-hemagglutinin (NTNH) proteins, which may cause adverse reactions such as coagulation and allergies.

[0005] The second method is to obtain botulinum toxin type A through genetic recombination technology, by constructing the botulinum toxin type A protein gene into an expression system through recombinant protein technology, and obtaining it through fermentation and purification. However, it is found from existing literature that it is difficult to obtain a product with the same amino acid structure as botulinum toxin type A produced by wild-type botulinum (Clostridium botulinum) through this technology. Therefore, the safety and effectiveness of the products obtained by the above method need to be re-evaluated in the long term.

[0006] Patent CN114989271A (Method for preparing recombinant botulinum toxin type A) uses genetic recombination technology to express the light chain protein and heavy chain protein of botulinum toxin type A respectively; the light chain protein is subjected to a first denaturation treatment to obtain a first denatured product; the heavy chain protein is subjected to a second denaturation treatment to obtain a second denatured product; the first denatured product and the second denatured product are mixed in proportion, and renatured and assembled to obtain the BoNT / A. This method for preparing BoNT / A requires additional denaturation and renaturation treatments, which will produce disulfide bond mismatches. The process steps are relatively cumbersome and complicated, and the yield is not high. Moreover, the first amino acid at the N-terminus of the light chain and the heavy chain is methionine (M), which is inconsistent with the amino acid structure of BoNT / A obtained by extracting and preparing through wild-type Clostridium botulinum culture.

[0007] Patent CN114957482B (A modified single-chain polypeptide of neurotoxin and its use) and patent CN118006523A (Recombinant genetically engineered bacteria of botulinum toxin type A and its preparation and use) also use genetic engineering recombinant technology to prepare botulinum toxin type A, but their amino acid structures are inconsistent with the botulinum toxin type A obtained by culturing and extracting wild-type Clostridium botulinum. More specifically, the naturally extracted botulinum toxin type A is as follows: the sequence of the heavy chain N-terminus (HC-N) is ALNDLCIK, the sequence of the light chain N-terminus (LC-N) is PFVNKQFN, and the sequence of the light chain C-terminus (LC-C) is LLCVRGIITSK. In patent CN118006523A, the above sequences are as follows: the sequence of the heavy chain N-terminus (HC-N) is GSK ALNDLCIK, the sequence of the light chain N-terminal (LC-N) is GS PFVNKQFN, the sequence of the light chain C-terminus (LC-C) is LLCVRGIITSK TKSLVPR . It can be seen that the amino acid sequence of the recombinant botulinum toxin type A in the prior art is actually different from that of the naturally extracted botulinum toxin type A. In addition, the naturally extracted botulinum toxin type A mentioned here refers to commercial products already on the market, including but not limited to Botox (https: / / www.genome.jp / entry / D08957) and Xeomin (https: / / www.pmda.go.jp / drugs / 2020 / P20200703001 / ). For detailed N-terminal and C-terminal sequence alignment, please see Figure 1 , Figure 2 and Figure 3 It can be seen that the existing recombinant expression technology is still unable to obtain botulinum toxin type A with the same structure as natural extraction.

[0008] The inconsistency between the amino acid sequence and the naturally extracted botulinum toxin type A products on the market means that the product has certain safety risks and requires sufficient experimental research to achieve product safety certification, which greatly prolongs the product development cycle. Although the traditional extraction method can obtain botulinum toxin type A with a natural structure, the process cost and time consumption are not ideal. It is urgent to develop a method for preparing botulinum toxin type A based on genetic engineering recombinant technology to improve the safety of botulinum toxin type A products and the simplicity and stability of its production process without changing the protein structure of natural botulinum toxin type A. Summary of the invention

[0009] The purpose of the present invention is to provide a recombinant single-chain protein of botulinum toxin type A to solve the technical problem that the preparation process of the existing botulinum toxin type A with natural structure is complicated and difficult to meet the application requirements. The botulinum toxin type A prepared by genetic engineering recombinant technology adopted in this patent has high safety, simple and stable process, and the amino acid structure is consistent with the amino acid sequence obtained by culture extraction of wild-type Clostridium botulinum.

[0010] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0011] A single-chain protein of recombinant botulinum toxin type A, comprising: BoNT / A-LC—linker—BoNT / A-HC, wherein:

[0012] (1) BoNT / A-LC is botulinum toxin type A light chain or a modified botulinum toxin type A light chain;

[0013] (2) Linker is the connection region; the connection region includes a connection peptide, a tag protein polypeptide, a connection peptide and a protease recognition site in sequence;

[0014] (3) BoNT / A-HC is the heavy chain of botulinum toxin type A.

[0015] Furthermore, the amino acid sequence of the botulinum toxin type A light chain is shown in SEQ ID NO.1; the amino acid sequence of the modified botulinum toxin type A light chain is shown in SEQ ID NO.2; and the amino acid sequence of the botulinum toxin type A heavy chain is shown in SEQ ID NO.3.

[0016] Furthermore, the structure of the connection area is as follows:

[0017] (Connector Peptide) a —(Tagged protein peptide) b —(Connector Peptide) c - protease recognition site;

[0018] Among them, a is 1-10; b is 1-2; c is 1-10.

[0019] Further, the sequence of the connecting peptide is GGGGS, GGGS, GGS, GGGQ or GEQP;

[0020] The tag protein polypeptide is selected from a histidine tag; preferably, the sequence of the histidine tag is selected from HHHHHH or HQHQHQ.

[0021] Further, the protease used to cleave the protease recognition site is trypsin or Lys-C enzyme, preferably Lys-C enzyme; the sequence of the protease recognition site is X d Y; wherein X is any amino acid except K and R, Y is K or R, and d is 0-10.

[0022] Further, the recombinant botulinum toxin type A is obtained by enzymatic cleavage of the single-chain protein of the recombinant botulinum toxin type A with Lys-C or trypsin;

[0023] The recombinant botulinum toxin type A has the same amino acid sequence as Botox and Xeomin extracted and prepared from wild strains of Clostridium botulinum;

[0024] The sequence of the light chain of the recombinant botulinum toxin type A is shown in SEQ ID NO.11, and the sequence of the heavy chain of the recombinant botulinum toxin type A is shown in SEQ ID NO.3;

[0025] Among them, the cysteine ​​at position 429 of SEQ ID NO.11 and the cysteine ​​at position 6 of SEQ ID NO.3 form a disulfide bond; the cysteine ​​at position 787 of SEQ ID NO.3 and the cysteine ​​at position 832 of SEQ ID NO.3 form a disulfide bond.

[0026] The technical solution also provides a method for preparing botulinum toxin type A by recombinant expression, characterized in that a single-chain protein of recombinant botulinum toxin type A is expressed by a microbial expression system; preferably, the microbial expression system is a prokaryotic expression system; preferably, the prokaryotic organism is Escherichia coli;

[0027] Preferably, the strain of Escherichia coli includes but is not limited to BL21(DE3), BL21(DE3)plysS, JM109(DE3), and Rosetta(DE3); more preferably, the strain of Escherichia coli is BL21(DE3).

[0028] Furthermore, an expression vector is transferred into the microbial expression system, wherein the expression vector is formed by integrating a nucleotide fragment of a single-chain protein of botulinum toxin type A into an empty vector, and the empty vector includes but is not limited to pET-26b, pET-30a, and pET-22a vectors; after fermentation expression in the microbial expression system and bacterial cell disruption, a supernatant containing the target protein is obtained; the supernatant is purified by affinity column, cation exchange chromatography, enzyme digestion, and cation exchange chromatography to obtain high-quality recombinant botulinum toxin type A.

[0029] The technical solution also provides a method of using the gene recombination technology of the solution for the preparation of other types of botulinum toxins.

[0030] The technical solution also provides a use of a recombinant botulinum toxin type A in the preparation of a drug for treating a nervous system disease, or in the preparation of a drug for treating a muscle spasm disease, or in the preparation of a drug for relieving pain, or in the preparation of a beauty product.

[0031] The technical solution also provides a gene encoding a single-chain protein of botulinum toxin type A.

[0032] The technical solution also provides an engineered cell that expresses a single-chain protein of botulinum toxin type A.

[0033] The technical principle of this technical solution is:

[0034] In this technical solution, a single-chain protein sequence is designed using gene recombination technology, optimized by cDNA, and expressed in Escherichia coli. The newly designed sequence (BoNT / A single-chain polypeptide) includes, in sequence, a first functional region (light chain) containing a metal ion-dependent protease activity domain, a connecting region, and a second functional region (heavy chain) containing a binding domain and a translocation domain. The connecting region is between the light and heavy chains. By a single enzyme cut, the connecting region can be removed, and the heavy chain and the light chain form a BoNT / A protein. In addition, the structure of the BoNT / A protein obtained in this solution is consistent with the amino acid structure of the naturally extracted BoNT / A protein. Specifically, the protein sequences of the light chain and heavy chain of the formed BoNT / A protein are completely consistent with the natural sequence, and no redundant amino acid residues appear at the N-terminus and C-terminus. A disulfide bond consistent with the natural BoNT / A protein is formed between the light chain and the heavy chain. Although there are existing methods for engineering cells to express BoNT / A single-chain polypeptides, due to sequence design issues, the finished BoNT / A protein usually has certain enzyme cleavage site sequences or residues of connecting short peptides, that is, the final product is not a product that is completely consistent with the amino acid structure of natural BoNT / A protein. The sequence design of this solution and the use of specific proteases for enzyme cleavage ensure that the connecting region can be completely removed by enzyme cleavage to form light chains and heavy chains that are consistent with the natural amino acid structure.

[0035] This patent uses a constructed strain to produce botulinum toxin type A, and high-quality recombinant botulinum toxin type A is obtained after single-chain protease cleavage and purification. The single-chain protein is expressed in Escherichia coli, and the resulting bacteria are sequentially subjected to bacterial lysis, Ni column chromatography, cation chromatography, enzyme cleavage, cation chromatography and anion chromatography to obtain a high-purity BoNT / A protein. The above multiple steps can complete the process preparation in 2-3 days, which improves the production efficiency of BoNT / A protein.

[0036] The beneficial effects of this solution are:

[0037] (1) Use genetic recombination expression technology to prepare botulinum toxin type A to improve safety.

[0038] (2) Obtain botulinum toxin type A with a structure and amino acid sequence consistent with natural extraction.

[0039] (3) The preparation process is simple, the cycle is short, and the product quality is high and uniform. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a comparison diagram of the N-terminal sequence of the botulinum toxin type A light chain (the result of comparison of the extracted natural botulinum toxin type A BoNT / A sequence, the recombinant botulinum toxin type A sequence obtained from CN114957482B, and the recombinant botulinum toxin type A sequence obtained from CN118006523A).

[0041] Figure 2 This is a comparison diagram of the C-terminal sequence of the botulinum toxin type A light chain (the result of comparison of the extracted natural botulinum toxin type A BoNT / A sequence, the recombinant botulinum toxin type A sequence obtained from CN114957482B, and the recombinant botulinum toxin type A sequence obtained from CN118006523A).

[0042] Figure 3 This is a comparison diagram of the N-terminal sequence of the heavy chain of botulinum toxin type A (the result of comparison of the extracted natural botulinum toxin type A BoNT / A sequence, the recombinant botulinum toxin type A sequence obtained from CN114957482B, and the recombinant botulinum toxin type A sequence obtained from CN118006523A).

[0043] Figure 4 Schematic diagram of the plasmid vector map and connection process of Example 1.

[0044] Figure 5 This is the SDS PAGE electrophoresis image of Example 2.

[0045] Figure 6 This is the complete molecular weight UV annotation diagram of the test product in Example 3.

[0046] Figure 7This is the deconvoluted intact molecular weight histogram of the test product in Example 3.

[0047] Figure 8 This is the UV annotation image of the test sample in Example 3 after reduction.

[0048] Fig. 9 This is the deconvoluted molecular weight column chart of the light chain of the test sample after reduction in Example 3.

[0049] Fig.10 This is the deconvoluted molecular weight column chart of the heavy chain of the test sample in Example 3 after reduction.

[0050] Fig.11 The test peptide segment of Example 3 (1:K12&:NEM(2)[6 37]+H + ) Primary and secondary mass spectrometry identification spectra.

[0051] Fig.12 The test peptide segment (2:K25&:NEM[5]+H + ) Primary and secondary mass spectrometry identification spectra.

[0052] Fig.13 The test peptide segment (2:K38&:NEM

[16] +H + ) Primary and secondary mass spectrometry identification spectra.

[0053] Fig.14 The test peptide segment (2:K44&:NEM[4]+H + ) Primary and secondary mass spectrometry identification spectra.

[0054] Fig.15 This is the BPI spectrum of the Lys-C enzymatic hydrolysis product detected by mass spectrometry in Example 3.

[0055] Fig.16 This is the component plot mass spectrum of the C-terminal peptide of the light chain of the test sample Lys-C digested in Example 3.

[0056] Fig.17 This is the primary identification mass spectrum of the C-terminal peptide segment of the light chain of the test sample Lys-C cleavage in Example 3.

[0057] Fig.18 This is the secondary identification mass spectrum of the C-terminal peptide segment of the light chain of the test sample Lys-C cleavage in Example 3.

[0058] Fig.19 This is the component plot mass spectrum of the Lys-C cleaved heavy chain C-terminal peptide of the test sample in Example 3.

[0059] Fig. 20This is the primary identification mass spectrum of the Lys-C-digested heavy chain C-terminal peptide of the test sample in Example 3.

[0060] Fig.21 This is the secondary identification mass spectrum of the C-terminal peptide segment of the heavy chain of the test sample Lys-C digested in Example 3.

[0061] Fig. 22 It is the near-ultraviolet CD spectrum of the test product of Example 3.

[0062] Fig.23 This is the far-ultraviolet CD spectrum of the test product in Example 3.

[0063] Fig.24 This is the DAS score reference chart of Example 5. DETAILED DESCRIPTION

[0064] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0065] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below.

[0066] The term "tag protein" refers to a type of protein molecule that can bind to a specific ligand.

[0067] The terms "BoNT / A," "BoNT / A protein," and "BoNT / A component" all refer to botulinum toxin type A.

[0068] The terms "light chain", "LC", "BoNT / A-LC" and "BoNT / A-LC protein" all refer to the light chain protein of botulinum toxin type A. The light chain is the part responsible for enzymatic activity and is a zinc-dependent metalloprotease that can specifically cleave the SNARE protein complex on the presynaptic membrane of the nerve, preventing the release of acetylcholine, thereby causing muscle relaxation and paralysis.

[0069] The terms "heavy chain", "HC", "BoNT / A-HC" and "BoNT / A-HC protein" all refer to the heavy chain protein of botulinum toxin type A. The heavy chain is divided into two functional regions. The N-terminal part is responsible for binding to receptors on the surface of nerve cells and mediating the entry of the toxin into the cell; the C-terminal part helps the toxin pass through the cell membrane during endocytosis and releases the light chain into the cytoplasm.

[0070] The terms "BoNT / A single-chain protein" and "Botulinum toxin type A single-chain protein" refer to a single-chain polypeptide comprising a heavy chain and a light chain, which is expressed by an engineered bacterium. Subsequently, through events such as proteolytic cleavage, the heavy chain and the light chain are separated into two polypeptide chains, which are bonded by disulfide bonds to form "BoNT / A protein".

[0071] The terms "BoNT / A protein" and "botulinum toxin type A protein" refer to botulinum toxin type A extracted from Clostridium botulinum, which is a protein formed by enzymatic cleavage of polypeptides and the connection of heavy and light chains through disulfide bonds. The heavy chain sequence is the same as SEQ ID NO.3 below, and the light chain sequence is the sequence formed by removing the C-terminal decapeptide and N-terminal methionine M from SEQ ID NO.1 below (SEQ ID NO.11).

[0072] The term "Lys-C enzyme" refers to a protease that can specifically hydrolyze peptide bonds at the carboxyl end of lysine (Lys, K), cleaving protein chains at the carboxyl end following the lysine residue.

[0073] The term "cation exchange chromatography" refers to a separation technique that exploits the electrostatic interaction between positively charged molecules and negatively charged functional groups on the stationary phase. It is primarily used to separate molecules that are positively charged at a given pH, such as certain proteins, peptides, or other biomacromolecules.

[0074] The term "anion exchange chromatography" refers to a separation technique based on the interaction of negatively charged molecules with positively charged ligands on a stationary phase. This chromatographic method is mainly used to separate proteins, peptides and other biomolecules with dissociable negative charges. At certain pH values, these molecules acquire a negative charge that allows electrostatic attraction to the positively charged groups on the stationary phase.

[0075] The term "affinity chromatography" refers to a chromatographic technique based on specific interactions between biomolecules. This method exploits the high affinity and specific binding between two molecules, such as the interaction between an antibody and its antigen, an enzyme and its cofactor, a receptor and its ligand, etc. By selecting an appropriate ligand and immobilizing it on the solid support of the chromatography column, highly specific enrichment and purification of a specific target molecule can be achieved.

[0076] The term "engineered cells" refers to cells that have been modified through genetic engineering technology. These cells can be bacteria, yeast, mammalian cells or even insect cells, designed to achieve specific biological functions or produce specific biological products.

[0077] The term "engineered bacteria" refers to microorganisms, usually bacteria, that have been modified through genetic engineering technology to achieve specific functions or produce specific products.

[0078] The term "competent cells" refers to cells that have been specially treated so that they can absorb and integrate foreign DNA under natural conditions.

[0079] The following is further described in detail through specific implementation methods:

[0080] Example 1: Design and construction of recombinant BoNT / A expression plasmid

[0081] The structure of natural BoNT / A protein is divided into two parts: light chain (LC) and heavy chain (HC). The light chain and heavy chain are linked by a pair of disulfide bonds (formed by two cysteines at the C-terminus of the light chain and the N-terminus of the heavy chain), and the heavy chain has a pair of disulfide bonds (formed by two cysteines at the C-terminus of the heavy chain). The light chain is an active domain with zinc-dependent metalloendopeptidase activity, which is the toxic part of the toxin; the heavy chain contains two domains, a binding domain and a translocation domain. The binding domain is responsible for binding to the corresponding receptor on the nerve cell membrane and forming an ion channel on the endosomal membrane. The translocation domain is responsible for the translocation of the light chain and transfers the light chain into the cell. When designing an expression vector, the heavy chain and light chain of BoNT / A are usually integrated into the same expression vector at the same time. After the fusion protein is expressed, the heavy chain and light chain are separated by enzyme cleavage, and the two are in the correct conformation of disulfide bond connection. In this technical solution, a connecting peptide is inserted between the fusion protein of the light chain and the heavy chain to form a new BoNT / A single-chain protein.

[0082] The light chain of the BoNT / A single-chain protein is called the first functional region containing the metal ion-dependent protease activity domain. Its sequence can be a full-length sequence or a truncated sequence (the native sequence without the C-terminal TKSLDKGYNK). The full-length sequence of the light chain of the BoNT / A protein is shown in SEQ ID NO.1 (the gray highlighted cysteine ​​residue C in the sequence is an amino acid residue for forming a disulfide bond, the same below; the 430th cysteine ​​of SEQ ID NO.1 or 2 and the 6th cysteine ​​of SEQ ID NO.3 form a disulfide bond):

[0083]

[0084] The modified botulinum toxin type A light chain sequence is shown in SEQ ID NO.2:

[0085]

[0086] The amino acid sequences of SEQ ID NO.1 and SEQ ID NO.2 can be designed with corresponding nucleotide sequences according to the degeneracy of codons to adapt to the expression requirements of different engineering bacteria, as long as the sequence of the translation product is SEQ ID NO.1 or SEQ ID NO.2.

[0087] The nucleotide sequence corresponding to SEQ ID NO.1 is SEQ ID NO.4:

[0088]

[0089] The nucleotide sequence corresponding to SEQ ID NO.2 is SEQ ID NO.5:

[0090]

[0091] The heavy chain of the natural BoNT / A protein is called the second functional region containing the binding domain and the translocation domain. The sequence of the heavy chain of botulinum toxin type A is shown in SEQ ID NO.3 (the gray highlighted cysteine ​​residue C in the sequence is an amino acid residue for forming a disulfide bond: the cysteine ​​residue at position 787 of SEQ ID NO.3 and the cysteine ​​residue at position 832 of SEQ ID NO.3 form a disulfide bond):

[0092] ALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL。

[0093] The amino acid sequence of SEQ ID NO.3 can be designed with a corresponding nucleotide sequence according to the degeneracy of the codons to adapt to the expression requirements of different engineered bacteria, as long as the sequence of the translation product is SEQ ID NO.3. In the subsequent experiments of this technical solution, the nucleotide sequence corresponding to SEQ ID NO.3 used specifically is (SEQ ID NO.6):

[0094]

[0095] The heavy chain and light chain are connected by a connecting peptide, and the amino acid sequence of the connecting peptide is:

[0096] (Connector Peptide) a (Tagged protein peptide) b (Connector Peptide) c X d Y; wherein a: 1-10; b: 1-2; c: 1-10; d: 0-10. That is, in the linker sequence (also called linker), there are GS short peptide (glycine-serine, i.e., linker peptide), histidine tag, GS short peptide, enzyme cleavage related sequence (X d Y). Preferably, the sequence of the GS short peptide is selected from GGGGS, GGGS, GGS, GGGQ or GEQP; preferably, the label protein polypeptide adopts a histidine tag, and the histidine tag is selected from HHHHHH or HQHQHQ. The enzyme used in this scheme is Lys-C enzyme or trypsin (preferably Lys-C enzyme), in X d The cleavage is performed at the carboxyl end of Y of Y. Wherein, X is any amino acid except K and R, Y is K or R, and the value range of d is preferably 0-3, more preferably 0-1.

[0097] The sequence of the single-chain protein of the recombinant botulinum toxin type A BoNT / A is: BoNT / A-LC—linker—BoNT / A-HC, which are connected end to end. BoNT / A-LC is a botulinum toxin type A light chain or a modified botulinum toxin type A light chain; linker is a connecting region; the connecting region includes a connecting peptide, a tag protein polypeptide, a connecting peptide and a protease recognition site in sequence (see above for details); BoNT / A-HC is a botulinum toxin type A heavy chain. Single-chain proteins that conform to the aforementioned "BoNT / A-LC—linker—BoNT / A-HC" structure can be constructed through expression vectors, expression bacteria, protein expression, chromatography purification, Lys-C enzyme or trypsin digestion, and chromatography purification (the preparation process is described later in this example) to form a botulinum toxin BoNT / A with an amino acid sequence consistent with the natural structure. That is, the heavy chain and light chain sequences of the formed botulinum toxin product are consistent with the commercial products Botox and Xeomin. In addition, for the single-chain protein conforming to the aforementioned "BoNT / A-LC—linker—BoNT / A-HC" structure, during the enzymatic cleavage process, the amount of Lys-C enzyme required can be as low as 0.05 mg of Lys-C enzyme per 1 g of BoNT / A single-chain protein; and the residual amount of Lys-C enzyme in the prepared botulinum toxin product is less than 0.1 ng / mg (Lys-C enzyme mass / botulinum toxin protein mass).

[0098] In the following examples, the single-chain polypeptide shown in SEQ ID NO.7 is specifically used for experimental description. The sequence of SEQ ID NO.7 is specifically:

[0099] BoNT / A-LC(SEQ ID NO.1)-GGGGSGGGGSGGGGSHHHHHHGGGGSGGGGSGGGGSPK-BoNT / A-HC(SEQ ID NO.3)

[0100] In the subsequent specific embodiments, the nucleotide sequence of the connecting peptide is shown in SEQ ID NO.8:

[0101] GGCGGAGGTGGCTCTGGAGGCGGTGGATCTGGTGGTGGCGGATCACCACCAC CATCACCACGGTGGAGGCGGGAGCGGAGGCGGCGGTAGTGGTGGAGGAGGCTCTCCC AAG.

[0102] The nucleotide fragment of BoNT / A single-chain protein is added with restriction sites at both ends by conventional means in the prior art, and then integrated into the multiple cloning site of the empty expression vector (appropriate restriction sites are selected as needed), and the expression vector expressing the BoNT / A protein precursor is transferred into competent cells to form engineered cells (engineered bacteria). The BoNT / A single-chain protein can be obtained through protein expression in the engineered cells, which is a conventional operation method in the prior art. In the subsequent experimental operation, HindⅢ and Nde I restriction sites are inserted at both ends of the nucleotide fragment of BoNT / A single-chain protein, and then the nucleotide fragment of BoNT / A single-chain protein is integrated into the commercial pET-26b empty expression vector through HindⅢ and Nde I restriction sites by conventional restriction and ligation methods in the prior art, that is, the expression vector (pET-26b-BoNT / A) expressing the BoNT / A single-chain protein is obtained. For details of the plasmid map and the schematic diagram of the ligation process, see Figure 4 . The expression vector is transferred into Escherichia coli, and an engineered bacterium expressing recombinant BoNT / A protein (BoNT / A protein precursor) is obtained through conventional transformation and screening. The specific engineered bacterium selected for subsequent experimental studies is Escherichia coli BL21 (DE3). Escherichia coli strains include but are not limited to BL21 (DE3), BL21 (DE3) plysS, JM109 (DE3), and Rosetta (DE3); more preferably, the strain of Escherichia coli is BL21 (DE3). The above methods are all conventional molecular cloning methods (recombinant technologies) in the prior art, and can be found in reference books such as "Molecular Cloning Experiment Guide", which will not be repeated here.

[0103] Example 2: Recombinant BoNT / A expression and protein purification

[0104] The recombinant BoNT / A engineered bacteria were inoculated into a shake flask filled with LB medium for culture. When OD600 = 1.6-2.5, they were transferred to a 5L fermenter for culture. The initial culture volume was 2L, the culture temperature was 37°C, the stirring speed was 300-800rpm, and when the OD600 value rose to 20-25, induction was started. The inducer was isopropyl-β-D-thiogalactoside (IPTG), the concentration was 0.5mM, and the induction time was 3-20h. The fermentation broth was examined for bacterial growth under a microscope. After 3-20h of induction, the fermentation broth was collected and centrifuged at 4°C and 7000rpm for 10min to collect the bacterial cells.

[0105] The obtained bacteria were sequentially subjected to bacterial disruption, Ni column chromatography, cation chromatography, enzyme digestion and cation chromatography. Except for the specific Lys-C enzyme used in the enzyme digestion step, the other processes were conventional processes for extracting and purifying proteins from bacteria in the prior art. The specific processing process is described as follows:

[0106] (1) Bacterial cell collection: The fermented bacterial liquid was centrifuged at 7500 r / min for 10 min in a refrigerated centrifuge to collect the bacterial cells, which were then frozen at -20°C.

[0107] (2) Dispersion and disruption of bacteria: Add 100 g / L of bacteria into lysis solution (PBS) and stir to suspend. When there are no large particles, ultrasonic disruption is performed. Centrifuge in a refrigerated centrifuge at 7500 r / min, 10 min, and 4°C to collect the supernatant.

[0108] (3) Ni column purification:

[0109] The reagents used included: balancing solution A: 20 mM sodium dihydrogen phosphate, pH 7.0; and elution solution B: 20 ​​mM sodium dihydrogen phosphate, 0.3 M imidazole, pH 7.0.

[0110] The supernatant collected by centrifugation was loaded onto a pre-equilibrated Ni column (Monmix MC60-NTA Ni), and after re-equilibration with equilibration solution A, conventional elution was performed using elution solution B to collect the target peak containing the BoNT / A single-chain protein to obtain the Ni column purified product. Ni column purification is mainly to separate the fusion protein with a histidine tag (BoNT / A single-chain protein) from other proteins.

[0111] (4) Cation exchange chromatography:

[0112] The reagents used include: balancing solution A: 20 mM sodium dihydrogen phosphate, pH 6.5; elution solution B: 20 ​​mM sodium dihydrogen phosphate, 0.5 M sodium chloride, pH 6.5.

[0113] The Ni column purified product obtained by Ni column recovery is loaded onto a pre-equilibrated 30S column (BestPoly 30S), and after re-equilibration with equilibration solution A, conventional elution is performed using elution solution B to collect the target peak containing BoNT / A single-chain protein to obtain the first ion exchange purified product. The target protein and other impurities are further separated by cation exchange chromatography.

[0114] (5) Enzyme digestion:

[0115] The pH of the cation exchange chromatography sample was adjusted to 8.0-8.5, and 0.05-1 mg of Lys-C enzyme was added to 1 g of BoNT / A single-chain protein for enzymatic digestion (i.e., the mass ratio of Lys-C enzyme to BoNT / A single-chain protein was controlled at 1:1000-1:20000), and the enzymatic digestion was carried out at 20-25° C. for 2-24 h.

[0116] Preferably, the enzyme cleavage conditions are: pH 8.0, the usage ratio of BoNT / A single-chain protein and Lys-C enzyme is 1 g:1 mg, the enzyme cleavage time is 4 h, and the enzyme cleavage temperature is 25°C.

[0117] The BoNT / A single-chain protein is cut into two parts, heavy chain and light chain, by enzyme cleavage. The sequences of the heavy chain and light chain are consistent with the sequences of the light chain and heavy chain in the natural BoNT / A protein double chain, and the intermediate linking peptide is removed by enzyme cleavage. The heavy chain and light chain automatically form a BoNT / A protein with the correct conformation. The sequence design and enzyme cleavage method of the BoNT / A single-chain protein in this scheme are used, and the enzyme cleavage position is "……TSK↓ TKSLDKGYNK …”, “…GGGGSPK↓ALNDL”. The linking region “GGGGSGGGGSGGGGSHHHHHHGGGGSGGGGSGGGGSPK” can be completely removed by enzyme cleavage, and the decapeptide “TKSLDKGYNK” at the C-terminus of the first functional region (light chain) can be removed. The sequences of the remaining light chain and heavy chain are consistent with those of the natural BoNT / A protein. In this scheme, enzyme cleavage occurs at two specific lysines of the BoNT / A single-chain protein. Although the BoNT / A single-chain protein also has other lysine sites, a specific structure is formed at the above two lysine sites (relative to the incorrect structure of the natural protein), which is easy for Lys-C enzyme to recognize and cleave, and enzyme cleavage does not occur at other lysine sites, thereby ensuring that the correct heavy chain and light chain sequences are formed by cleavage. Therefore, the upstream design of the BoNT / A single-chain protein in this scheme is different from the prior art and can ensure that the BoNT / A protein with a natural structure is obtained.

[0118] (6) Cation exchange chromatography:

[0119] The reagents used include: balancing solution A: 20 mM sodium dihydrogen phosphate, pH 6.5; elution solution B: 20 ​​mM sodium dihydrogen phosphate, 0.5 M sodium chloride, pH 6.5.

[0120] After the digested sample is diluted, the pH is adjusted to 6.5 and loaded onto a pre-equilibrated 30S column (BestPoly 30S). After re-equilibration with equilibration solution A, conventional elution is performed using elution solution B to collect the target peak containing BoNT / A protein. Non-related proteins are further removed by cation exchange chromatography in this step. The collected product is the final product of BoNT / A, i.e., the stock solution. The eluted sample is subjected to SDS-PAGE electrophoresis, as shown in FIG. Figure 5 As shown. Figure 5 In A, from left to right, lane 1 is the non-reduced single-chain protein before enzyme cleavage; lane 2 is the reduced single-chain protein before enzyme cleavage; lane 3 is the marker; lane 4 is the non-reduced stock solution (BoNT / A protein); lane 5 is the reduced stock solution (BoNT / A protein separated into heavy chain and light chain after reduction treatment). Figure 5 B is the SDS-PAGE electrophoresis result of the final stock solution. From left to right, lane 4 is the stock solution after non-reduction treatment (BoNT / A protein); lane 2 is the marker; lane 3 is the stock solution after reduction treatment (BoNT / A protein separated into heavy chain and light chain after reduction treatment). Elisa was used to detect the residual Lys-C in the stock solution prepared in this example, and the residual amount was less than 0.1 ng / mg (Lys-C enzyme mass / botulinum toxin protein mass).

[0121] Example 3: Characterization of recombinant BoNT / A protein

[0122] (1) Intact molecular weight analysis

[0123] Mass spectrometry conditions: The sample was analyzed by mass spectrometry using a XevoG2-XS QTof mass spectrometer. Analysis time: 15 min.

[0124] Detection method: positive ion, parent ion scanning range: 500-4000m / z.

[0125] The test samples were separated by ultra-high performance liquid chromatography system. Liquid A was an aqueous solution containing 0.1% formic acid, and liquid B was an acetonitrile solution containing 0.1% formic acid. The chromatographic column was balanced with liquid A. The test samples were loaded by an automatic sampler and then separated by the chromatographic column at a flow rate of 0.3 mL / min, a detection wavelength of 280 nm, and a column temperature of 80°C.

[0126] After LC-MS detection and software analysis, the molecular weight of the intact protein was measured to be 148172.9Da. For the UV-annotated diagram of the intact molecular weight of the test sample, see Figure 6 ; The deconvoluted intact molecular weight histogram of the test sample is shown in Figure 7 .

[0127] (2) Reduced molecular weight analysis

[0128] Take an appropriate amount of the test sample into the denaturing buffer 6M guanidine hydrochloride solution, add DTT with a final concentration of 100mM, and incubate at 100℃ for 5 minutes. The test sample is loaded by an automatic sampler and then separated by a chromatographic column at a flow rate of 0.3mL / min, a detection wavelength of 280nm, and a column temperature of 80℃.

[0129] After LC-MS detection and software analysis, the molecular weight of the light chain protein after reduction was measured to be 50024.4Da, and the molecular weight of the heavy chain was 98151.6Da. Figure 8 ; The deconvoluted intact molecular weight histogram of the light chain of the test sample after reduction can be found at Fig. 9 The deconvoluted intact molecular weight histogram of the heavy chain of the test sample after reduction can be found at Fig.10 .

[0130] Chinese patent CN114957482B describes the situation of natural BoNT / A protein: the natural BoNT / A light chain has a natural ring region at its C-terminus. Under natural conditions, the ring will be cleaved by a Clostridial endopeptidase that has not yet been identified when the toxin is released from the Clostridium during cell lysis. For BoNT / A, the ring sequence on its light chain can be cleaved into a decapeptide (i.e., the aforementioned TKSLDKGYNK) during cleavage. The cleaved light chain and heavy chain are connected by a disulfide bond at the N-terminus and C-terminus by a cysteine ​​residue respectively.

[0131] The sequence of the light chain in the natural active BoNT / A product (dimer formed by heavy chain and light chain) is actually SEQ ID NO.1 minus the TKSLDKGYNK sequence at the C-terminus and the M at the N-terminus (E. coli has its own methionine aminopeptidase. After the protein is expressed in E. coli, the initial M is automatically removed without any other operation), and the theoretical molecular weight is about 50024 Da (i.e., SEQ ID NO.11, see below). The theoretical molecular weight of the heavy chain of natural BoNT / A is about 98151 Da (i.e., SEQ ID NO.3).

[0132] Among them, naturally extracted botulinum toxin type A refers to the commercial products Botox and Xeomin available on the market.

[0133] For more information about Botox, please visit https: / / www.genome.jp / entry / D08957;

[0134] For more information about Xeomin, please visit https: / / www.pmda.go.jp / drugs / 2020 / P20200703001 / .

[0135] The heavy chain sequence of naturally extracted botulinum toxin type A (the light chain and heavy chain dimer formed after the single-chain protein is cleaved) is shown in SEQ ID NO.3, and the light chain sequence is shown in SEQ ID NO.11:

[0136] PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTS IVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVT LAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSG KFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK.

[0137] The molecular weight of the obtained BoNT / A light chain is consistent with that of the naturally extracted BoNT / A double-chain light chain through expression plasmid construction, engineered bacterial expression, enzyme digestion and purification treatment; the molecular weight of the obtained BoNT / A heavy chain is basically consistent with that of the naturally extracted BoNT / A double-chain heavy chain. Through the single-chain protein sequence design and enzyme digestion operation of this scheme, the connecting peptide between the heavy chain and the light chain is completely removed, and the light chain and heavy chain of BoNT / A consistent with the naturally extracted structure are obtained.

[0138] (3) Disulfide bond analysis and free thiol analysis

[0139] After denaturation and NEM alkylation, replace with 20mM PB buffer. Take part of it and add Lys-C, and enzymatically react at 37℃ for 18h. Acidify with 7M Gdn-HCl to terminate the reaction, take part of the enzymatic hydrolysis product, add 1M TCEP to reduce. The enzymatic hydrolysis product of the test sample is separated by liquid phase system.

[0140] The enzymatic hydrolysis products of the test samples were desalted and separated by high performance liquid chromatography and then detected by high resolution mass spectrometry. Analysis time: 120 min, detection method: positive ion (MSE), parent ion scanning range: 300-2000 m / z.

[0141] After non-reductive enzymatic hydrolysis and liquid quality detection analysis, two pairs of disulfide bonds were identified that were consistent with the theoretical disulfide bond pairing mode, namely C429-C443 and C1224-C1269. From the perspective of the finished BoNT / A product (the light chain and heavy chain have been cut and bound by disulfide bonds), the 429th amino acid of its light chain is cysteine, and the 6th amino acid of its heavy chain is cysteine, and a disulfide bond is formed between the two (L C429 and H C6); the 787th amino acid of its heavy chain is cysteine, and the 832nd amino acid is cysteine, and a disulfide bond is formed between the two (H C787 and H C832). The formation of disulfide bonds is consistent with that of naturally extracted BONT / A.

[0142] The test sample was lyophilized and concentrated and diluted with 20mM PB solution. After denaturation and NEM alkylation, it was replaced with 20mM PB buffer. A portion of it was added with Lys-C and the enzymatic reaction was carried out at 37℃ for 18h. The reaction was terminated by acidification with 7M Gdn-HCl, and a portion of the enzymatic hydrolysis product was taken and reduced by adding 1M TCEP. The enzymatic hydrolysis product of the test sample was desalted and separated by high-performance liquid chromatography and then detected by scanning mass spectrometry analysis using a high-resolution mass spectrometer. Analysis time: 120min, detection mode: positive ion (MSE), parent ion scanning range: 300-2000m / z. Mass spectrometry acquisition data was retrieved by UNIFI (Waters) software for theoretical free thiol peptide data retrieval and post-reduction verification analysis.

[0143] The identification results of the alkylated peptides of the test samples are shown in Table 1, and the free thiol ratios of the test samples are shown in Table 2. + ) The primary and secondary mass spectrometry identification spectra are shown in Fig.11 , the test peptide (2:K25&:NEM[5]+H + ) The primary and secondary mass spectrometry identification spectra are shown in Fig.12 , the test peptide (2:K38&:NEM

[16] +H + ) The primary and secondary mass spectrometry identification spectra are shown in Fig.13 , the test peptide (2:K44&:NEM[4]+H + ) The primary and secondary mass spectrometry identification spectra are shown in Fig.14 .

[0144] Table 1: Identification results of alkylated modified peptides of the test sample (including: observed retention time (Observed RT), expected mass (Expected mass), observed mass (Observed mass), mass deviation (Mass Error) and other items)

[0145]

[0146] Table 2: Free thiol ratio of the test samples

[0147] name Free thiol ratio% <![CDATA[1:K12&:NEM(2)[6 37]+H + ]]> 100 <![CDATA[2:K25&:NEM[5]+H + ]]> 100 <![CDATA[2:K38&:NEM

[16] +H + ]]> 100 <![CDATA[2:K44&:NEM[4]+H + ]]> 100

[0148] After analysis, the test sample was alkylated with NEM for free thiol groups and then enzymatically cleaved and reduced. Liquid chromatography-mass spectrometry showed that cysteine ​​mainly existed in a free state, with contents of C133: 100%, C164: 100%, C780: 100%, C956: 100%, and C1049: 100%.

[0149] (4) N-terminal sequence analysis

[0150] Electrophoresis: Take enough sample and add reducing sample buffer, mix well and boil, load the sample into two wells, add pre-stained marker. Power on the concentrated gel at 100V for 15min, and power on the separation gel at 180V for 30min.

[0151] Transfer: Prepare a PVDF membrane, cut it into the same size as the gel, and evenly wet it in 100% methanol. Place the gel on a thick filter paper that has also been soaked in the transfer buffer, then cover the PVDF membrane on the gel, and then cover it with a soaked thick filter paper, expel the air to make it full of liquid, and finally clamp them together in the electrotransfer tank. Place the transfer clip in the transfer tank with the side with the gel facing the negative end, add transfer buffer, turn on the external circulation to cool to 10°C, and turn on the magnetic stirrer under the electrotransfer tank, adjusting it to a low speed. Turn on the power, wet transfer, and power on at 250mA for 90 minutes. After the power is turned on, take out the transfer clip, remove the PVDF membrane and dye it with dye solution, and then rinse it with purified water until the bands are clear. Scan the photo with EPSON and upload it, and cut the desired bands and dry the test sample.

[0152] Computer test:

[0153] Place the cut PVDF membrane into the reactor. After assembling the reactor, place it in a fixed position on the instrument. Set the test product name, number, test cycle number and method file through the PPSQ Analysis software to start the N-terminal test.

[0154] Data and graph processing:

[0155] The raw data and spectra generated by PPSQ are identified by PPSQ DataProcessing software and the corresponding spectra are exported.

[0156] After the above analysis, the N-terminal sequencing results of the test sample are:

[0157] Heavy chain: NH2-Ala-Leu-Asn-Asp-Leu-X-Ile-Lys-Val-Asn-Asn-Trp-Asp-Leu-Phe.

[0158] (ALNDLXIKVNNWDLF, Cys corresponds to X, which is consistent with the theoretical sequence)

[0159] Light chain: NH2-Pro-Phe-Val-Asn-Lys-Gln-Phe-Asn-Tyr-Lys-Asp-Pro-Val-Asn-Gly.

[0160] (PFVNKQFNYKDPVNG)

[0161] It can be seen that the N-termini of the heavy chain and light chain of the BoNT / A prepared by this solution are consistent with the sequence of the naturally extracted BoNT / A, and no extra amino acid residues are inserted or deleted. Using this technical solution, the sequence of the N-terminus of the heavy chain is completely consistent with SEQ ID NO.3, and the sequence of the N-terminus of the light chain is completely consistent with SEQ ID NO.5, and there are no unexpected intermediates. Using the enzyme cleavage method of this solution, other amino acid residues that are not components of the natural heavy chain will not be introduced at the N-terminus of the heavy chain.

[0162] (5) C-terminal analysis

[0163] Enzymatic hydrolysis of test sample: Take the freeze-dried concentrated sample and dilute it with 20mM PB solution, add an equal volume of 8M UA, denature at 37℃ for 1h. Add Lys-C, enzymatic hydrolysis at 37℃ for 18h, acidify with 7M Gdn-HCl to terminate the reaction, and add 1M TCEP for reduction.

[0164] LC-MS / MS identification: The enzymatic samples were separated using an UPLC liquid phase system (ACQUITY UPLC I-Class).

[0165] After separation, a high-resolution mass spectrometer was used for detection and scanning mass spectrometry analysis. Analysis time: 120 min, detection method: positive ion (MSE), parent ion scanning range: 300-2000 m / z.

[0166] Mass spectrometry data processing: Mass spectrometry data were processed using UNIFI control program (1.9.4, Waters), and the theoretical sequence of the test sample was selected as the database, and then the database matching search was performed. The peptide information obtained by Lys-C digestion of the test sample was retrieved from the theoretical sequence database and matched by the liquid mass spectrometry data software, and the C-terminal sequence peptides of the light chain and heavy chain were identified.

[0167] Lys-C enzymatic hydrolysis product mass spectrometry detection BPI spectrum see Fig.15 , the component plot of the C-terminal peptide of the light chain of the test sample Lys-C enzyme digestion can be found in Fig.16 The mass spectrum of the primary identification of the C-terminal peptide fragment of the light chain of the test sample Lys-C enzyme digestion can be found in Fig.17 The secondary identification mass spectrum of the C-terminal peptide fragment of the light chain of the test sample Lys-C enzyme digestion can be found in Fig.18 , the component plot of the C-terminal peptide of the Lys-C cleavage heavy chain of the test sample can be found in Fig.19 The mass spectrum of the primary identification of the C-terminal peptide fragment of the Lys-C cleavage heavy chain of the test sample can be found at Fig. 20 The secondary identification mass spectrum of the C-terminal peptide fragment of the Lys-C cleavage heavy chain of the test sample can be found in Fig.21 .

[0168] Based on the above experimental results, the C-terminal sequence of the light chain of the test sample LPJT-099 stock solution (batch number: 20240601) was identified as LLCVRGIITSK by mass spectrometry analysis;

[0169] The C-terminal sequence of the heavy chain is: LVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL.

[0170] The C-terminal sequences of the heavy and light chains are consistent with the theoretical ones, that is, the sequence of the BoNT / A protein prepared in this scheme is consistent with that of the natural BoNT / A protein (molecular weight, C-terminus and N-terminus are all consistent).

[0171] Based on the above molecular weight analysis, amino acid terminal analysis and disulfide bond analysis, it can be seen that the BoNT / A synthesized in this scheme is consistent with natural BoNT / A in terms of amino acid sequence (primary structure), light chain and heavy chain composition, disulfide bond formation, etc. Therefore, the preparation method of this technical solution can obtain a double-chain botulinum toxin BoNT / A that is consistent with the natural endotoxin extraction structure.

[0172] (6) Circular dichroism analysis

[0173] After the cuvette was soaked in 2M HNO3 overnight and rinsed with deionized water and dried, the background and blank buffers were scanned in sequence, and then an appropriate amount of the test sample was added to the cuvette for near-UV (250-340nm) and far-UV scanning (190-260nm). The near-UV CD spectrum of the test sample is detailed in Fig. 22 , the far-UV CD spectrum of the test sample is shown in Fig.23 .

[0174] Pro-Data Viewer software was used to perform averaging and smoothing on the scanned spectra, and the number of smoothing was set to 3. The ratio of the CD values ​​of the peaks and valleys of the standard was calculated, and the effective ratio range was 2.08±0.06. CDNN software was used to predict the secondary structure of the spectra.

[0175] CDNN software was used to fit and calculate the secondary structure of the test products, including helix, beta-pleated sheet (including antiparallel and parallel), beta-turn and random coil. The statistical results of the secondary structure analysis of each test product are shown in Table 3.

[0176] Table 3: Prediction statistics of secondary structures of each test product

[0177] Helix Antiparallel Parallel Beta-Turn Random Coil 19.4％ 28.7％ 6.4％ 18.1％ 29.2％

[0178] Example 4: Activity determination of recombinant BONT / A protein (toxicity of stock solution)

[0179] (1) Experimental design and grouping and drug administration:

[0180] After the adaptive feeding of 26-30 day old male Kunming mice (SPF grade), 60 mice with similar body weight were selected and randomly divided into 6 groups, 10 mice in each group. Samples were taken for the preparation of the test sample to be tested, and the samples were diluted to equal concentrations. After administration, the toxic reactions of the mice and the death of animals in each group were closely observed for 4 consecutive days. The sample is the sample prepared in Example 2, and the eluate obtained after the cation chromatography of "(6) cation exchange chromatography" is collected and referred to as the stock solution.

[0181] (2) Experimental process:

[0182] Before the experiment, the experimental mice were weighed and evenly distributed to each group according to their weight to ensure that there was no statistical difference in the average weight of each group of animals. The experiment was divided into 6 groups, with 10 males in each group. Intraperitoneal injection: 1 person extracted the test solution, and another person checked and completed the animal administration operation. After the injection was completed, the administration time was recorded. The grouping and dosage information of the test product botulinum toxin type A are shown in Table 4.

[0183] Under the experimental conditions, the LD of the recombinant botulinum toxin type A stock solution was calculated by SPSS. 50 The toxicity of the sample was 3.917 pg / piece, and the toxicity was converted to 2.55×10 8 LD 50 / mg, which is comparable to the toxicity of the wild-type BONT / A in the prior art.

[0184] Table 4: Test results of toxicity effect of BONT / A protein obtained in Example 2

[0185]

[0186]

[0187] Example 5: DAS Scoring

[0188] 40 healthy, sexually mature female SD rats (SPF grade), weighing about 180-200g. After the adaptive feeding, they were randomly divided into 4 groups (10 rats / group): vehicle control group, low-dose group, medium-dose group, and high-dose group. The samples were injected intramuscularly on one side. After the administration, the hind toe abduction (DAS) of the rats was closely observed and scored according to the scoring table (see the scoring criteria for details). Fig.24 , Reference: Ron S Broide, The rat Digit Abduction Score (DAS) assay: a physiological model for assessing botulinum neurotoxin-induced skeletal muscle paralysis, Toxicon.2013Sep:71:18-24.doi:10.1016 / j.toxicon.2013.05.004.Epub 2013May 23.).

[0189] Before the experiment, rats were evenly distributed to each group according to their weight to ensure that there was no statistical difference in the average weight of each group of animals. The samples were diluted to the corresponding concentrations. The experiment was divided into 4 groups: vehicle control group, low-dose group (5U / kg), medium-dose group (10U / kg), and high-dose group (20U / kg). For a single dose, the gastrocnemius muscle was selected for multiple injections on the unilateral hind limb of the animal; the injection volume at each point did not exceed 0.20mL. The animals were scored according to the scoring table before and after the drug at D1, D2, D3, D4, D5, D7, D9, D11, D15, D19, D23, D28, D35, and D42. The experimental dose and grouping information are shown in Table 5.

[0190] Table 5: Experimental groups and dosing arrangements

[0191]

[0192] The experimental results are shown in Table 6. The experimental results show that the application of BONT / A prepared in this scheme can effectively block neuromuscular conduction, inhibit muscle contraction, and achieve denervation.

[0193] Table 6: DAS scoring results of BONT / A protein obtained in Example 2

[0194]

[0195]

[0196] Comparative Example 1

[0197] In the prior art, endotoxins with natural structures are mainly obtained in two ways: extraction from Clostridium botulinum; and expression of the heavy chain and light chain in different engineered bacteria, respectively, followed by assembly.

[0198] In the prior art, attempts have also been made to integrate the heavy chain and light chain of botulinum toxin to form a fusion protein, express it in the same engineered bacteria, and then obtain botulinum toxin through enzyme cleavage and other operation steps. For example, in Chinese patent CN114957482B (a modified single-chain polypeptide of neurotoxin and its use), the technology connects the tag protein, light chain and heavy chain in sequence to form a fusion protein, and sets enzyme cleavage sites between the light chain and the heavy chain and between the tag protein and the light chain. Specifically, the botulinum toxin precursor sequence in this patented technology is: tag protein, first protease cleavage site LEVLFQGPL, connecting short peptide GS, light chain of the first functional amino acid structure region BoNT / A (C-terminus: ... CVRGIITS, when designing the sequence, the amino acid residues of the ring knot region of the C-terminus were removed in advance), second protease cleavage site LEVLFQGP and heavy chain of the second functional amino acid structure region BONT / A (N-terminus: ALNDLCIK ...). After the recognition sequence LEVLFQGP of the tobacco etch virus (TEV) protease is detected, it is cut (LEVLFQ↓GP) and then recombined to form a dimer of light chain and heavy chain (double-chain botulinum toxin). However, the above method cannot obtain a botulinum toxin with a natural structure. For example: the light chain sequence is not a natural sequence, and it still has residues of the enzyme cleavage site at its C-terminus, and it still has residues of the enzyme cleavage site and a GS short peptide at its N-terminus. For another example, its heavy chain is also not a natural sequence, and there are also residual amino acid residues of the enzyme cleavage site at the N-terminus of its heavy chain. Since the sequence of the botulinum toxin obtained by this patented technology contains amino acid residues of non-natural botulinum toxin, the botulinum toxin obtained by this method is not a botulinum toxin with a natural structure, and is different from the present technical solution. For sequence alignment, please refer to Figure 1-3 .

[0199] Chinese patent CN118006523A (Recombinant genetically engineered bacteria of botulinum toxin type A and its preparation and application) also attempted to produce recombinant botulinum toxin type A. The specific scheme is: use the pET-28a vector to integrate the recombinant single-chain polypeptide of botulinum toxin, then perform protein expression, cutting and purification, and finally form botulinum toxin type A. Among them, the recombinant single-chain polypeptide includes a gene fragment 1 expressing a GST tag, a gene fragment 2 expressing a thrombin recognition site, and a gene fragment 3 expressing botulinum toxin type A BoNT / A; the region connecting the BoNT / A light chain and the heavy chain includes the amino acid sequence of GST described in another thrombin recognition site. The thrombin recognition site is LVPRGS, and the cutting position is LVPR↓GS. Similarly, the N-terminus of the heavy chain of BONT / A formed by cutting according to the above method, or the N-terminus and C-terminus of the light chain, will retain some non-natural BONT / A amino acid residues, resulting in the overall product (especially the primary structure of the polypeptide) not being a natural structure. For sequence alignment, see Figure 1-3 .

[0200] The special BONT / A precursor (single-chain polypeptide) design of this solution can ensure that after enzyme cleavage, the heavy and light chains of BONT / A with the same structure as the natural extraction are obtained, and the overall product is consistent with the naturally extracted BoNT / A. Since naturally extracted BoNT / A has been widely used, its safety and effectiveness can be effectively guaranteed. The new BONT / A preparation method developed by this technical solution can not only improve the efficiency of the preparation process, but also ensure that the product structure is completely consistent with the natural extraction structure. Compared with the existing technology of preparing BoNT / A through single-chain polypeptides of recombinant proteins, it has more advantages.

[0201] Comparative Example 2

[0202] The prior art usually expresses the light and heavy chains separately, and then combines the light and heavy chains to form BoNT / A. For example, in Chinese patent CN115894641B (Construction of type A botulinum toxin mutants and genetically engineered bacteria thereof), the light and heavy chains were expressed separately, and the cysteines of the light and heavy chains were mutated. However, the expression of light and heavy chains separately will increase the process flow and complexity to a certain extent. In addition, the light and heavy chains in this scheme are mutated, and the structure of the obtained product is quite different from that of the naturally extracted product. Therefore, its safety and effectiveness still need further study. Compared with the product whose structure in this scheme is consistent with that of naturally extracted BoNT / A, it is more difficult and takes a longer period to realize the clinical application of this product.

[0203] Comparative Example 3: Study on the introduction position of restriction site

[0204] This comparative example has conducted a lot of exploration on the setting of the restriction site. First, an attempt was made to set the restriction site at the N-terminus of the single-chain protein. The amino acid sequence of the single-chain protein is as follows (SEQ ID NO.9):

[0205]

[0206]

[0207] In the above sequence, a histidine tag and a connecting sequence (shown by double underline) are added to the N-terminus of the light chain, and the light chain and the heavy chain are directly connected by a decapeptide, and a connecting peptide containing a histidine tag is not set between the two as in this scheme. An enzyme cleavage site "AEAEAPK↓PFVNKQFNY" is set at the N-terminus of the light chain, and it is planned to be cut here. After cutting, the N-terminus of the light chain is consistent with the natural sequence. BoNT / A was prepared according to the methods of Examples 1 and 2, except that the single-chain protein sequence was replaced with SEQID NO.9, and the enzyme cleavage method was completely performed in accordance with the preferred method of Example 2. The obtained botulinum toxin BoNT / A was sequenced at the N-terminus / C-terminus of the light chain and the heavy chain. The N-terminus of the light chain was SDKII (no cleavage was formed at the designed enzyme cleavage site), the N-terminus of the heavy chain was ALNDL (consistent with the natural sequence), and the C-terminus was consistent with the natural sequence. The sequencing results showed that it was difficult for the N-terminus of the light chain to form the expected enzyme cleavage method when the sequence was designed, and the structure of the obtained botulinum toxin was inconsistent with that of the natural botulinum toxin. Therefore, it is not appropriate to add a tag to the N-terminus of the single-chain protein.

[0208] Then, an attempt was made to set the restriction site at the C-terminus of the single-chain protein. The amino acid sequence of the single-chain protein is as follows (SEQ ID NO.10):

[0209]

[0210]

[0211] In the above sequence, a histidine tag and a connecting sequence (shown by double underline) are added to the C-terminus of the heavy chain, and the light chain and the heavy chain are directly connected by a decapeptide (in the natural form), and a connecting peptide containing a histidine tag is not set between the two as in this scheme. BoNT / A was prepared according to the methods of Examples 1 and 2, except that the single-chain protein sequence was replaced with SEQ ID NO.10, and the enzyme cleavage method was completely performed in accordance with the preferred method of Example 2. The molecular weights of the light chain and heavy chain of the obtained botulinum toxin BoNT / A were detected (reduced molecular weight), and the molecular weight of the light chain was 50025.8, and the molecular weight of the heavy chain existed in various situations: 98280.60, 95564.5, and 95039.0. The results showed that the light chain was consistent with the theory, and the heavy chain was not completely cut and miscut. The molecular weight detection results showed that according to the above-mentioned single-chain protein sequence design, the C-terminus of the heavy chain was difficult to form the expected enzyme cleavage method when the sequence was designed, and the structure of the obtained botulinum toxin was inconsistent with that of the natural botulinum toxin. Therefore, it is not appropriate to add a tag to the C-terminus of a single-chain protein.

[0212] This technical solution designs a histidine tag between the light chain and the heavy chain. The addition of the histidine tag facilitates protein purification. Through enzymatic cleavage with Lys-C enzyme, a botulinum toxin with a natural structure can be formed. Compared with setting the tag protein at the C-terminus and N-terminus of a single-chain protein, an unexpected technical effect is achieved.

[0213] Comparative Example 4: Study on enzyme cutting method

[0214] For the single-chain protein sequence of SEQ ID NO.10 in Comparative Example 3, the C-terminus of the heavy chain cannot be correctly cleaved by the enzyme cleavage method of Example 2, but the C-terminus of the light chain and the N-terminus of the heavy chain can be correctly cleaved. The connection between the heavy chain and the light chain of the single-chain protein in Comparative Example 3 is actually the same as that of the natural single-chain protein (the light chain is directly connected to the heavy chain through the aforementioned decapeptide).

[0215] More specifically, the enzyme digestion method for single-chain protein is: pH 8.0, the dosage ratio of BoNT / A single-chain protein and Lys-C enzyme is 1g:1mg (the ratio of enzyme to single-chain protein is 1:1000), the enzyme digestion time is 4h, and the enzyme digestion temperature is 25°C. Under this condition, the C-terminus of the light chain and the N-terminus of the heavy chain can be correctly cut (the obtained product is consistent with the natural sequence). However, the amount of Lys-C enzyme used in this enzyme digestion method is still relatively large, which does not take advantage of the saving of raw materials and the reduction of impurities in the final product. Try to reduce the amount of enzyme, and then observe the enzyme digestion results. The enzyme digestion method for the single-chain protein of SEQ ID NO.10 is modified as follows: pH 8.0, 1g BoNT / A single-chain protein is added with 0.05mg Lys-C enzyme for enzyme digestion (the ratio of enzyme to single-chain protein is 1:20000), and the enzyme digestion is carried out at 20°C for 16h. The remaining operation modes are the same as in Example 2, only the enzyme digestion conditions are changed, and the enzyme digestion products are obtained by preparing the stock solution in the manner of Example 2. The protein in the stock solution is detected, and it is found that the heavy chain and light chain of a large number of proteins are not cut, which is specifically embodied in: the protein reduction treatment in the stock solution is followed by electrophoresis, and the electrophoresis results show that in addition to the light chain band and the heavy chain band, a band with a larger molecular weight (about the sum of the molecular weights of the light chain and the heavy chain) also appears, indicating that the light chain and the heavy chain are not fully cut under the enzyme digestion conditions. In addition, the molecular weight is reduced, and in addition to the correct light chain molecular weight (about 50024Da) and heavy chain molecular weight (about 98151Da), the reduced molecular weight of the light chain and the heavy chain all presents a variety of forms, or slightly larger than the correct molecular weight or slightly less than the correct molecular weight. It is explained that the natural connection mode is adopted between the light chain and the heavy chain, and it is desired to realize the correct cutting of the C-terminal of the light chain and the N-terminal of the heavy chain, and to form a sequence that conforms to the natural structure, there are certain requirements for the enzyme digestion mode (especially the amount of Lys-C enzyme).

[0216] The enzymatic cleavage method was studied for the single-chain protein sequence (SEQ ID NO.7) of this scheme. The enzymatic cleavage method was specifically as follows: pH 8.0, 1g BoNT / A single-chain protein was added with 0.05mg of Lys-C enzyme for enzymatic cleavage (the ratio of enzyme to single-chain protein was 1:20000), and the enzymatic cleavage was carried out at 20°C for 16h. The rest of the operation was the same as in Example 2, only the enzymatic cleavage conditions were changed, and the enzymatic cleavage product was obtained by preparing the stock solution in accordance with the method of Example 2. The reduced molecular weight was measured, and the result was consistent with the theory, light chain: about 50024.00, heavy chain: about 98151.75, and no other molecular weight forms were presented. There was no situation where the light chain and the heavy chain were not completely cleaved, and there was no situation where the cleavage site of the C-terminus of the light chain and the N-terminus of the heavy chain was wrong. The sequences of the light chain and heavy chain of the obtained botulinum toxin product were consistent with the natural sequence, and the cleavage effect of increasing the amount of Lys-C enzyme in Example 2 was consistent, and a botulinum toxin product with a structure consistent with the natural structure could be formed. The Lys-C residue in the stock solution prepared by this preparation method was detected by Elisa, and the residual amount was less than 0.1 ng / mg (Lys-C enzyme mass / botulinum toxin protein mass).

[0217] According to the above experimental results, this solution sets a connecting peptide with a histidine tag between the light chain and the heavy chain. Due to the presence of the histidine tag, the subsequent purification process becomes more effective. The presence of the connecting peptide ensures that the correct cut is formed between the light chain and the heavy chain, and the C-terminus of the light chain and the N-terminus of the heavy chain are completely consistent with their natural structure. And due to the presence of the connecting peptide, the amount of enzyme required for enzyme cutting is greatly reduced. Even when the ratio of enzyme to single-chain protein is 1:20000, the C-terminus of the light chain and the N-terminus of the heavy chain can also form a correct cutting form, and there will be no situation of insufficient enzyme cutting or wrong enzyme cutting site. Therefore, the upstream design of the botulinum toxin product of the present technical solution is different from the conventional method of the prior art. Through the design of the connecting peptide with a histidine tag between the light chain and the heavy chain, it is ensured that the sequence of the final product is completely consistent with the natural botulinum toxin, and it is also ensured that the amount of enzyme used can be greatly reduced when the single-chain polypeptide is cut, thereby reducing the reagent cost, reducing the impurity residues in the product, and reducing the difficulty of the subsequent purification process.

[0218] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A single-chain protein of recombinant botulinum toxin type A, characterized in that: The components include: BoNT / A-LC—linker—BoNT / A-HC, wherein: (1) BoNT / A-LC is botulinum toxin type A light chain or a modified botulinum toxin type A light chain; (2) Linker is the connection region; the connection region includes a connection peptide, a tag protein polypeptide, a connection peptide and a protease recognition site in sequence; (3) BoNT / A-HC is the heavy chain of botulinum toxin type A.

2. The single-chain protein of recombinant botulinum toxin type A according to claim 1, characterized in that: The amino acid sequence of the botulinum toxin type A light chain is shown in SEQ ID NO.1; the amino acid sequence of the modified botulinum toxin type A light chain is shown in SEQ ID NO.2; and the amino acid sequence of the botulinum toxin type A heavy chain is shown in SEQ ID NO.

3.

3. The single-chain protein of recombinant botulinum toxin type A according to claim 1, characterized in that: The structure of the connection area is as follows: (Connector Peptide) a —(Tagged protein peptide) b —(Connector Peptide) c - protease recognition site; Among them, a is 1-10; b is 1-2; c is 1-10.

4. The single-chain protein of recombinant botulinum toxin type A according to claim 2, characterized in that: The sequence of the connecting peptide is GGGGS, GGGS, GGS, GGGQ or GEQP; The tag protein polypeptide is selected from a histidine tag; preferably, the sequence of the histidine tag is selected from HHHHHH or HQHQHQ.

5. The single-chain protein of recombinant botulinum toxin type A according to claim 2, characterized in that: The protease used to cleave the protease recognition site is trypsin or Lys-C enzyme, preferably Lys-C enzyme; the sequence of the protease recognition site is X d Y; wherein X is any amino acid except K and R, Y is K or R, and d is 0-10.

6. A single-chain protein of a recombinant botulinum toxin type A according to any one of claims 1 to 5, characterized in that: Recombinant botulinum toxin type A obtained by enzymatic cleavage of the single-chain protein of recombinant botulinum toxin type A with Lys-C or trypsin; The amino acid sequence of the recombinant botulinum toxin type A is consistent with that of the botulinum toxin type A extracted and prepared from wild strains of Clostridium botulinum; The sequence of the light chain of the recombinant botulinum toxin type A is shown in SEQ ID NO.11, and the sequence of the heavy chain of the recombinant botulinum toxin type A is shown in SEQ ID NO.3; Among them, the cysteine ​​at position 429 of SEQ ID NO.11 and the cysteine ​​at position 6 of SEQ ID NO.3 form a disulfide bond; the cysteine ​​at position 787 of SEQ ID NO.3 and the cysteine ​​at position 832 of SEQ ID NO.3 form a disulfide bond.

7. A method for preparing botulinum toxin type A by recombinant expression, characterized in that: A microbial expression system is used to express the single-chain protein of the recombinant botulinum toxin type A according to any one of claims 1 to 5; preferably, the microbial expression system is a prokaryotic expression system; preferably, the prokaryotic organism is Escherichia coli; Preferably, the strain of Escherichia coli includes but is not limited to BL21(DE3), BL21(DE3)plysS, JM109(DE3), and Rosetta(DE3); more preferably, the strain of Escherichia coli is BL21(DE3).

8. The method for preparing botulinum toxin type A by recombinant expression according to claim 8, characterized in that: An expression vector is transferred into the microbial expression system, wherein the expression vector is formed by integrating a nucleotide fragment of a single-chain protein of type A botulinum toxin into an empty vector, and the empty vector includes but is not limited to pET-26b, pET-30a, and pET-22a vectors; after fermentation expression in the microbial expression system and bacterial cell disruption, a supernatant containing the target protein is obtained; the supernatant is purified by affinity column, cation exchange chromatography, enzyme digestion, and cation exchange chromatography to obtain high-quality recombinant type A botulinum toxin.

9. The method according to claim 7 is used for the preparation of other types of botulinum toxins.

10. Use of the recombinant botulinum toxin type A according to claim 5 in the preparation of a drug for treating a nervous system disease, or a drug for treating a muscle spasm disease, or a drug for relieving pain, or a cosmetic product.

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