Recombinant botulinum toxin and preparation method therefor

Abstract

The present invention relates to the technical field of a method for preparing a recombinant botulinum toxin type A, and particularly relates to a botulinum toxin type A obtained by means of gene recombination technology, the amino acid sequence of which is identical to the sequence of botulinum toxin type A prepared by means of extraction and purification from a wild Clostridium botulinum strain. Provided is a single-chain protein of botulinum toxin type A, sequentially comprising: a light chain of botulinum toxin type A or a modified light chain of botulinum toxin type A, a linking region, and a heavy chain of botulinum toxin type A. The single-chain protein is subjected to fermentation, crude purification, enzyme digestion, and fine purification to obtain high-quality recombinant botulinum toxin type A. The present invention can solve the existing problems of complex process, long period, high safety risk and the like in the preparation of botulinum toxin type A by using wild Clostridium botulinum strains, and also solve the existing problem that the amino acid sequence of botulinum toxin type A prepared by means of gene recombination technology cannot be identical to that of botulinum toxin type A prepared by wild Clostridium botulinum strains.

Description

Recombinant botulinum toxin and its preparation method Technical Field

[0001] This invention relates to the technical field of recombinant botulinum toxin preparation methods, specifically to type A botulinum toxin obtained by gene recombination technology, whose amino acid sequence is consistent with the amino acid sequence of type A botulinum toxin prepared by extraction and purification from wild-type strain Clostridium botulinum. Background Technology

[0002] Natural botulinum toxin type A (BoNT / A) is a potent neurotoxin produced by wild-type Clostridium botulinum and is commonly used in the medical field. Botulinum toxin type A primarily works by blocking neurotransmitter transmission and inhibiting muscle contraction to improve conditions such as facial spasms, cerebral palsy, blepharospasm, and cervical dystonia. It is also used to treat certain types of headaches, hyperhidrosis, and endocrine disorders.

[0003] In the existing technology, there are mainly two methods for preparation:

[0004] One method involves extraction from wild-type Clostridium botulinum, which is currently the primary method for producing type A botulinum toxin products on the market, such as Botox, Xeomin, and Dysport. However, preparing type A botulinum toxin using Clostridium botulinum is extremely demanding due to safety concerns, requiring highly sophisticated laboratory procedures and processes. Furthermore, this method is lengthy and complex. Wild-type Clostridium botulinum also produces hemagglutinin (HA) and non-toxic non-hemagglutinin (NTNH) proteins, which may cause adverse reactions such as hemocoagulation and allergic reactions.

[0005] Secondly, botulinum toxin type A can be obtained through gene recombination technology. This involves constructing the botulinum toxin type A protein gene into an expression system using recombinant protein technology, followed by fermentation and purification. However, existing literature indicates that it is difficult to obtain a product with an amino acid structure identical to that produced by wild-type Clostridium botulinum using this technology. Therefore, the safety and efficacy of products obtained by the above methods require re-evaluation and long-term assessment.

[0006] Patent CN114989271A (Preparation Method of Recombinant Type A Botulinum Toxin) employs gene recombination technology to express the light chain and heavy chain proteins of type A botulinum toxin, respectively. The light chain protein undergoes a first denaturation treatment to obtain a first denatured product; the heavy chain protein undergoes a second denaturation treatment to obtain a second denatured product; the first and second denatured products are mixed in a certain proportion and subjected to renaturation and assembly processes to obtain BoNT / A. This method for preparing BoNT / A requires additional denaturation and renaturation treatments, which can lead to disulfide bond mismatches. The process is relatively cumbersome and complex, resulting in a low yield. Furthermore, the first amino acid at the N-terminus of both the light and heavy chains is methionine (M), which is inconsistent with the amino acid structure of BoNT / A obtained through extraction from wild-type Clostridium botulinum.

[0007] Patents CN114957482B (A modified neurotoxin single-chain polypeptide and its use) and CN118006523A (Recombinant genetically engineered bacteria for type A botulinum toxin and its preparation and application) also use recombinant genetic engineering technology to prepare type A botulinum toxin, but their amino acid structures are inconsistent with those obtained by extracting and culturing wild-type Clostridium botulinum. More specifically, the naturally extracted type A botulinum toxin has the following sequence: the N-terminal sequence of the heavy chain (HC-N) is ALNDLCIK, the N-terminal sequence of the light chain (LC-N) is PFVNKQFN, and the C-terminal sequence of the light chain (LC-C) is LLCVRGIITSK. In patent CN118006523A, the sequence is as follows: the N-terminal (HC-N) sequence of the heavy chain is GSKALNDLCIK, the N-terminal (LC-N) sequence of the light chain is GSPFVNKQFN, and the C-terminal (LC-C) sequence of the light chain is LLCVRGIITSKTKSLVPR. It is evident that the amino acid sequence of the recombinant type A botulinum toxin in the prior art differs from that of naturally extracted type A botulinum toxin. Furthermore, the naturally extracted type A botulinum toxin mentioned here refers to commercially available products, 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 alignments, please refer to Figures 1, 2, and 3. It is clear that current recombinant expression techniques cannot yet obtain type A botulinum toxin with the same structure as naturally extracted botulinum toxin.

[0008] The inconsistency between the amino acid sequence and that of commercially available type A botulinum toxin products derived from natural sources indicates a certain safety risk. Extensive experimental research is needed to determine the product's safety, significantly extending the product development cycle. While traditional extraction methods can yield type A botulinum toxin with a natural structure, the process costs and time consumption are unsatisfactory. There is an urgent need to develop a method for preparing type A botulinum toxin based on genetic engineering recombination technology. This method should improve the safety of type A botulinum toxin products, as well as the simplicity and stability of its production process, without altering the protein structure of natural type A botulinum toxin. Summary of the Invention

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

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

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

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

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

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

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

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

[0017] (Connecting peptide) a —(Tag protein polypeptide) b — (Connecting peptide) c —Protein recognition site;

[0018] Where a is 1-10; b is 1-2; c is 1-10.

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

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

[0021] Furthermore, 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; where X is any amino acid except K and R, Y is K or R, and d is 0-10.

[0022] Furthermore, recombinant botulinum toxin type A was obtained by digesting the single-chain protein of recombinant botulinum toxin type A with Lys-C or trypsin.

[0023] The recombinant type A botulinum toxin has the same amino acid sequence as Botox and Xeomin, which were prepared from wild-type Clostridium botulinum.

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

[0025] Specifically, 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] This technical solution also provides a method for preparing type A botulinum toxin through recombinant expression, characterized in that a microbial expression system is used to express the single-chain protein of recombinant type A botulinum toxin; preferably, the microbial expression system is a prokaryotic expression system; preferably, the prokaryote 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 introduced into the microbial expression system. The expression vector is formed by integrating a nucleotide fragment of the single-chain protein of botulinum toxin type A into an empty vector. The empty vector includes, but is not limited to, pET-26b, pET-30a, and pET-22a vectors. After fermentation expression and cell disruption in the microbial expression system, a supernatant containing the target protein is obtained. The supernatant is purified by affinity column chromatography, cation exchange chromatography, enzyme digestion, and cation exchange chromatography to obtain high-quality recombinant botulinum toxin type A.

[0029] This technical solution also provides a method for using the gene recombination technology of this solution to prepare other types of botulinum toxin.

[0030] This technical solution also provides the application of recombinant type A botulinum toxin in the preparation of drugs for treating nervous system diseases, drugs for treating muscle spasm diseases, drugs for relieving pain, or cosmetic products.

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

[0032] This 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 as follows:

[0034] In this technical solution, a single-chain protein sequence designed using gene recombination technology is expressed in *E. coli* through cDNA optimization. The newly designed sequence (BoNT / A single-chain polypeptide) sequentially includes a first functional region (light chain) containing a metal-dependent protease active domain, a linker region, and a second functional region (heavy chain) containing both binding and translocation domains. The linker region is located between the light and heavy chains. This linker region can be removed by a single enzyme digestion, allowing the heavy and light chains to form the BoNT / A protein. Furthermore, the structure of the BoNT / A protein obtained in this solution is identical to the amino acid structure of the naturally extracted BoNT / A protein. Specifically, the protein sequences of the light and heavy chains of the formed BoNT / A protein are completely identical to the natural sequence, and no extra amino acid residues appear at the N-terminus or C-terminus. Disulfide bonds, identical to those in the natural BoNT / A protein, are formed between the light and heavy chains. While existing technologies allow for the expression of BoNT / A single-chain polypeptides using engineered cells, sequence design issues often result in residual enzymatic cleavage sites or linker peptides in the final BoNT / A protein product. This means the final product is not entirely identical to the amino acid structure of the natural BoNT / A protein. Our proposed method, through sequence design and the use of specific proteases for cleavage, ensures the complete removal of the linker region, resulting in light and heavy chains with a structure identical to the natural amino acid.

[0035] This patent utilizes a constructed bacterial strain for the production of botulinum toxin type A. After single-chain protease digestion and purification, high-quality recombinant botulinum toxin type A is obtained. The single-chain protein is expressed in *E. coli*, and the resulting bacterial cells are sequentially subjected to lysis, Ni column chromatography, cation chromatography, enzyme digestion, cation chromatography, and anion chromatography to obtain high-purity BoNT / A protein. The above multiple steps can be completed in 2-3 days, improving the production efficiency of BoNT / A protein.

[0036] The beneficial effects of this plan are as follows:

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

[0038] (2) Obtain type A botulinum toxin with the same structure and amino acid sequence as the naturally extracted botulinum toxin.

[0039] (3) The preparation process is simple and the cycle is short, resulting in high and uniform product quality. The purpose of this invention is to provide a single-chain protein of recombinant type A botulinum toxin to solve the technical problem that the existing preparation process of type A botulinum toxin with natural structure is complex and difficult to meet application requirements. The type A botulinum toxin prepared by the genetic engineering recombination technology used in this patent has high safety, simple and stable process, and its amino acid structure is consistent with the amino acid sequence obtained by culturing and extracting wild-type Clostridium botulinum.

[0040] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0041] A single-chain protein of recombinant botulinum toxin type A, comprising, in sequence: BoNT / A-LC—linker—BoNT / A-HC, wherein,

[0042] (1) BoNT / A-LC is a type A botulinum toxin light chain or a modified type A botulinum toxin light chain;

[0043] (2) The linker is the linker region; the linker region includes the linker peptide, the tag protein polypeptide, the linker peptide and the protease recognition site in sequence;

[0044] (3) BoNT / A-HC is a type A botulinum toxin heavy chain.

[0045] Furthermore, the amino acid sequence of the type A botulinum toxin light chain is shown in SEQ ID NO.1; the amino acid sequence of the modified type A botulinum toxin light chain is shown in SEQ ID NO.2; and the amino acid sequence of the type A botulinum toxin heavy chain is shown in SEQ ID NO.3.

[0046] Furthermore, the structure of the connection region is as follows:

[0047] (Connecting peptide) a —(Tag protein polypeptide) b — (Connecting peptide) c —Protein recognition site;

[0048] Where a is 1-10; b is 1-2; c is 1-10.

[0049] Furthermore, the sequence of the linker peptide is GGGGS, GGGS, GGS, GGGQ or GEQP;

[0050] The tagged protein polypeptide is selected from histidine tags; preferably, the sequence of the histidine tag is selected from HHHHHH or HQHQHQ.

[0051] Furthermore, 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; where X is any amino acid except K and R, Y is K or R, and d is 0-10.

[0052] Furthermore, recombinant botulinum toxin type A was obtained by digesting the single-chain protein of recombinant botulinum toxin type A with Lys-C or trypsin.

[0053] The recombinant type A botulinum toxin has the same amino acid sequence as Botox and Xeomin, which were prepared from wild-type Clostridium botulinum.

[0054] The sequence of the light chain of recombinant botulinum toxin type A is shown in SEQ ID NO.11, and the sequence of the heavy chain of recombinant botulinum toxin type A is shown in SEQ ID NO.3;

[0055] Specifically, 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.

[0056] This technical solution also provides a method for preparing type A botulinum toxin through recombinant expression, characterized in that a microbial expression system is used to express the single-chain protein of recombinant type A botulinum toxin; preferably, the microbial expression system is a prokaryotic expression system; preferably, the prokaryote is Escherichia coli;

[0057] 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).

[0058] Furthermore, an expression vector is introduced into the microbial expression system. The expression vector is formed by integrating a nucleotide fragment of the single-chain protein of botulinum toxin type A into an empty vector. The empty vector includes, but is not limited to, pET-26b, pET-30a, and pET-22a vectors. After fermentation expression and cell disruption in the microbial expression system, a supernatant containing the target protein is obtained. The supernatant is purified by affinity column chromatography, cation exchange chromatography, enzyme digestion, and cation exchange chromatography to obtain high-quality recombinant botulinum toxin type A.

[0059] This technical solution also provides a method for using the gene recombination technology of this solution to prepare other types of botulinum toxin.

[0060] This technical solution also provides the application of recombinant type A botulinum toxin in the preparation of drugs for treating nervous system diseases, drugs for treating muscle spasm diseases, drugs for relieving pain, or cosmetic products.

[0061] This technical solution also provides a gene encoding a single-chain protein of botulinum toxin type A.

[0062] This technical solution also provides an engineered cell that expresses a single-chain protein of botulinum toxin type A.

[0063] The technical principle of this technical solution is as follows:

[0064] In this technical solution, a single-chain protein sequence designed using gene recombination technology is expressed in *E. coli* through cDNA optimization. The newly designed sequence (BoNT / A single-chain polypeptide) sequentially includes a first functional region (light chain) containing a metal-dependent protease active domain, a linker region, and a second functional region (heavy chain) containing both binding and translocation domains. The linker region is located between the light and heavy chains. This linker region can be removed by a single enzyme digestion, allowing the heavy and light chains to form the BoNT / A protein. Furthermore, the structure of the BoNT / A protein obtained in this solution is identical to the amino acid structure of the naturally extracted BoNT / A protein. Specifically, the protein sequences of the light and heavy chains of the formed BoNT / A protein are completely identical to the natural sequence, and no extra amino acid residues appear at the N-terminus or C-terminus. Disulfide bonds, identical to those in the natural BoNT / A protein, are formed between the light and heavy chains. While existing technologies allow for the expression of BoNT / A single-chain polypeptides using engineered cells, sequence design issues often result in residual enzymatic cleavage sites or linker peptides in the final BoNT / A protein product. This means the final product is not entirely identical to the amino acid structure of the natural BoNT / A protein. Our proposed method, through sequence design and the use of specific proteases for cleavage, ensures the complete removal of the linker region, resulting in light and heavy chains with a structure identical to the natural amino acid.

[0065] This patent utilizes a constructed bacterial strain for the production of botulinum toxin type A. After single-chain protease digestion and purification, high-quality recombinant botulinum toxin type A is obtained. The single-chain protein is expressed in *E. coli*, and the resulting bacterial cells are sequentially subjected to lysis, Ni column chromatography, cation chromatography, enzyme digestion, cation chromatography, and anion chromatography to obtain high-purity BoNT / A protein. The above multiple steps can be completed in 2-3 days, improving the production efficiency of BoNT / A protein.

[0066] The beneficial effects of this plan are as follows:

[0067] (1) Use gene recombination expression technology to prepare type A botulinum toxin to improve safety.

[0068] (2) Obtain type A botulinum toxin with the same structure and amino acid sequence as the naturally extracted botulinum toxin.

[0069] (3) The preparation process is simple and the cycle is short, and the product quality is high and uniform. Attached Figure Description

[0070] Figure 1 shows the N-terminal sequence alignment of the light chain of botulinum toxin type A (the result of aligning 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).

[0071] Figure 2 shows the C-terminal sequence alignment of the light chain of botulinum toxin type A (the result of aligning 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).

[0072] Figure 3 shows the N-terminal sequence alignment of the heavy chain of botulinum toxin type A (the result of aligning 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).

[0073] Figure 4 shows the plasmid vector map and ligation process of Example 1.

[0074] Figure 5 shows the SDS-PAGE electrophoresis image of Example 2.

[0075] Figure 6 is a UV-coded diagram showing the complete molecular weight of the test sample in Example 3.

[0076] Figure 7 is a deconvolutioned complete molecular weight column chart of the test sample of Example 3.

[0077] Figure 8 shows the UV labeling diagram of the sample after reduction in Example 3.

[0078] Figure 9 is a bar chart of the deconvolution molecular weight of the light chain of the test sample after reduction in Example 3.

[0079] Figure 10 is a bar graph of the deconvolution molecular weight of the heavy chain after reduction of the test sample in Example 3.

[0080] Figure 11 shows the primary and secondary mass spectrometry identification spectra of the test peptide (1:K12&:NEM (2)

[0637] +H⁺) in Example 3.

[0081] Figure 12 shows the primary and secondary mass spectrometry identification spectra of the test peptide (2:K25&:NEM [5]+H⁺) in Example 3.

[0082] Figure 13 shows the primary and secondary mass spectrometry identification spectra of the test peptide (2:K38&:NEM

[0016] +H⁺) in Example 3.

[0083] Figure 14 shows the primary and secondary mass spectrometry identification spectra of the test peptide (2:K44&:NEM [4]+H⁺) in Example 3.

[0084] Figure 15 shows the BPI spectrum of the Lys-C enzymatic hydrolysis product from Example 3.

[0085] Figure 16 is the component plot mass spectrum of the C-terminal peptide of the Lys-C cleaved light chain of the test sample in Example 3.

[0086] Figure 17 shows the mass spectrum of the Lys-C enzyme-digested light chain C-terminal peptide of the test sample in Example 3 for primary identification.

[0087] Figure 18 shows the mass spectrometry spectrum of the secondary identification of the C-terminal peptide of the Lys-C cleaved light chain of the test sample in Example 3.

[0088] Figure 19 shows the component plot of the C-terminal peptide of the Lys-C cleaved heavy chain of the test sample in Example 3.

[0089] Figure 20 shows the mass spectrum of the Lys-C enzyme-digested heavy chain C-terminal peptide of the test sample in Example 3 for primary identification.

[0090] Figure 21 shows the mass spectrometry spectrum of the secondary identification of the C-terminal peptide of the Lys-C cleaved heavy chain of the test sample in Example 3.

[0091] Figure 22 shows the near-ultraviolet CD spectrum of the test sample in Example 3.

[0092] Figure 23 shows the far-ultraviolet CD spectrum of the test sample in Example 3.

[0093] Figure 24 is a reference chart of DAS score for Example 5. Embodiments of the present invention

[0094] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0095] To make the invention easier to understand, certain technical and scientific terms are defined below.

[0096] The term "tag protein" refers to a class of protein molecules that can bind to specific ligands.

[0097] The terms “BoNT / A,” “BoNT / A protein,” and “BoNT / A component” all refer to botulinum toxin type A.

[0098] 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 responsible for the enzyme activity and is a zinc-dependent metalloproteinase that can specifically cleave the SNARE protein complex on the presynaptic membrane of nerves, preventing the release of acetylcholine, thereby causing muscle relaxation and paralysis.

[0099] 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 portion is responsible for binding to receptors on the surface of nerve cells, mediating the entry of the toxin into the cell; the C-terminal portion helps the toxin cross the cell membrane during endocytosis, releasing the light chain into the cytoplasm.

[0100] The terms "BoNT / A single-chain protein" and "botulinum toxin type A single-chain protein" refer to single-chain polypeptides containing heavy and light chains, which are formed through expression in engineered bacteria. Subsequently, through events such as proteolytic cleavage, the heavy and light chains separate to form two polypeptide chains, which then bind together through disulfide bonds to form the "BoNT / A protein".

[0101] The terms "BoNT / A protein" and "botulinum toxin type A protein" refer to botulinum toxin type A extracted from Clostridium botulinum. It is a protein formed by linking the heavy and light chains via disulfide bonds after polypeptide cleavage. Its heavy chain sequence is the same as SEQ ID NO. 3 below, and its 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).

[0102] The term "Lys-C enzyme" refers to a protease that specifically hydrolyzes peptide bonds at the carboxyl terminus of lysine (Lys, K), cleaving the protein chain at the carboxyl terminus following the lysine residue.

[0103] The term "cation exchange chromatography" refers to a technique that separates molecules by utilizing the electrostatic interaction between positively charged molecules and negatively charged functional groups on a stationary phase. It is primarily used to separate molecules that are positively charged under given pH conditions, such as certain proteins, peptides, or other biomolecules.

[0104] The term "anion exchange chromatography" refers to a separation technique based on the interaction between negatively charged molecules and positively charged ligands on a stationary phase. This chromatographic method is primarily used to separate proteins, peptides, and other biomolecules carrying dissociable negative charges. At certain pH values, these molecules acquire a negative charge, allowing them to be electrostatically attracted to positively charged groups on the stationary phase.

[0105] The term "affinity chromatography" refers to a chromatographic technique based on specific interactions between biomolecules. This method utilizes the high affinity and specific binding between two molecules, such as the interaction between an antibody and its antigen, an enzyme and its cofactor, or a receptor and its ligand. By selecting appropriate ligands and immobilizing them on a solid support in the chromatographic column, highly specific enrichment and purification of specific target molecules can be achieved.

[0106] The term "engineered cell" refers to cells that have been modified through genetic engineering techniques. These cells can be bacteria, yeast, mammalian cells, or even insect cells, designed to achieve specific biological functions or produce specific biological products.

[0107] The term "engineered bacteria" refers to microorganisms, usually bacteria, that have been modified through genetic engineering techniques to achieve specific functions or produce specific products.

[0108] The term "competent cell" refers to a cell that, after special treatment, is able to absorb and integrate exogenous DNA under natural conditions.

[0109] The following detailed description illustrates the specific implementation method:

[0110] Example 1: Design and construction of recombinant BoNT / A expression plasmid

[0111] The natural BoNT / A protein consists of two parts: a light chain (LC) and a heavy chain (HC), linked by a disulfide bond (formed by two cysteine ​​residues at the C-terminus of the light chain and the N-terminus of the heavy chain). The heavy chain also contains a disulfide bond (formed by two cysteine ​​residues at the C-terminus of the heavy chain). The light chain is the active domain, possessing zinc-dependent metalloendopeptidase activity and representing the toxic portion of the toxin. The heavy chain contains two domains: a binding domain and a translocation domain. The binding domain binds to corresponding receptors on the nerve cell membrane and forms ion channels on the endosomal membrane. The translocation domain is responsible for the translocation of the light chain, transporting it into the cell. Typically, the BoNT / A heavy and light chains are integrated into the same expression vector. After expression, the heavy and light chains are separated by enzymatic cleavage, forming the correct conformation where they are linked by disulfide bonds. In this technical approach, a linker peptide is inserted between the light and heavy chains of the fusion protein, forming a novel BoNT / A single-chain protein.

[0112] The light chain of the BoNT / A single-chain protein is called the first functional region containing a metal-dependent protease active domain. Its sequence can be either full-length or truncated (the natural sequence with the C-terminus TKSLDKGYNK removed). The full-length sequence of the BoNT / A protein light chain is shown in SEQ ID NO.1 (the gray highlighted cysteine ​​residues C in the sequence are amino acid residues used to form disulfide bonds, the same applies below; the cysteine ​​at position 430 in SEQ ID NO.1 or 2 and the cysteine ​​at position 6 in SEQ ID NO.3 form a disulfide bond):

[0113] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNK。

[0114] The modified light chain sequence of botulinum neurotoxin type A is shown in SEQ ID NO.2:

[0115] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK。

[0116] The amino acid sequences of SEQ ID NO.1 and SEQ ID NO.2 can be designed with corresponding nucleotide sequences based on the degeneracy of the codons to meet the expression needs of different engineered bacteria, as long as the sequence of the translation product is SEQ ID NO.1 or SEQ ID NO.2.

[0117] The nucleotide sequence corresponding to SEQ ID NO.1 is SEQ ID NO.4:

[0118]

[0119] The nucleotide sequence corresponding to SEQ ID NO.2 is SEQ ID NO.5:

[0120]

[0121] The heavy chain of the natural BoNT / A protein, referred to as the second functional region containing both binding and transposition domains, is shown in SEQ ID NO.3 (the gray highlighted cysteine ​​residue C in the sequence represents the amino acid residue used to form a disulfide bond: cysteine ​​at position 787 and cysteine ​​at position 832 of SEQ ID NO.3 form a disulfide bond):

[0122] ALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL。

[0123] The amino acid sequence of SEQ ID NO.3 can be used to design corresponding nucleotide sequences based on codon degeneracy to suit the expression needs of different engineered bacteria, as long as the sequence of the translation product is SEQ ID NO.3. In subsequent experiments of this technical solution, the nucleotide sequence corresponding to SEQ ID NO.3 used is (SEQ ID NO.6):

[0124]

[0125] The heavy chain and light chain are linked by a linker peptide, the amino acid sequence of which is:

[0126] (Connecting peptide) a (Tag protein peptide) b (Connecting peptide) c X d Y; where a: 1-10; b: 1-2; c: 1-10; d: 0-10. That is, this linker sequence (also called the linker) sequentially contains a GS short peptide (glycine-serine, i.e., the linker peptide), a histidine tag, a GS short peptide, and an enzyme-related sequence (X). d Y). Preferably, the sequence of the GS short peptide is selected from GGGGS, GGGS, GGS, GGGQ, or GEQP; preferably, the tagged protein polypeptide uses a histidine tag, and the histidine tag is selected from HHHHHH or HQHQHQ. The enzyme used in this scheme is specifically Lys-C enzyme or trypsin (preferably Lys-C enzyme), in X d The carboxyl terminus of Y is cleaved. Here, X is any amino acid except K and R, Y is K or R, and the value of d is preferably 0-3, more preferably 0-1.

[0127] The sequence of the single-chain protein of recombinant botulinum toxin type A, BoNT / A, is: BoNT / A-LC—linker—BoNT / A-HC, linked together end-to-end. BoNT / A-LC is the light chain of botulinum toxin type A or a modified light chain of botulinum toxin type A; the linker is the linking region; the linking region sequentially includes a linker peptide, a tag protein polypeptide, a linker peptide, and a protease recognition site (see above for details); BoNT / A-HC is the heavy chain of botulinum toxin type A. Single-chain proteins conforming to the aforementioned "BoNT / A-LC—linker—BoNT / A-HC" structure can be processed through expression vector construction, expression bacteria construction, protein expression, chromatographic purification, enzymatic digestion with Lys-C enzyme or trypsin, and chromatographic purification (the preparation process is described later in this embodiment) to form botulinum toxin BoNT / A with an amino acid sequence consistent with the natural structure. That is, the heavy and light chain sequences of the resulting botulinum toxin product are consistent with the commercial products Botox and Xeomin. In addition, for single-chain proteins conforming to the aforementioned "BoNT / A-LC—linker—BoNT / A-HC" structure, the amount of Lys-C enzyme required during enzymatic digestion can be as low as 0.05 mg of Lys-C enzyme added to 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).

[0128] The following examples specifically illustrate the experiments using the single-chain polypeptide with the sequence shown in SEQ ID NO.7. The sequence of SEQ ID NO.7 is as follows:

[0129] BoNT / A-LC (SEQ ID NO.1)-GGGGSGGGGSGGGGSHHHHHHGGGGSGGGGSGGGGSPK- BoNT / A-HC (SEQ ID NO.3)

[0130] In the following specific embodiments, the nucleotide sequence of the linker peptide is shown in SEQ ID NO.8:

[0131] GGCGGAGGTGGCTCTGGAGGCGGTGGATCTGGTGGTGGCGGATCACACCACCACCATCACCACGGTGGAGGCGGGAGCGGAGGCGGCGGTAGTGGTGGAGGAGGCTCTCCCAAG.

[0132] The nucleotide fragment of the BoNT / A single-chain protein was inserted at both ends using conventional techniques, with restriction enzyme sites added as needed. This was then integrated into the multiple cloning site of an empty expression vector (appropriate restriction enzyme sites were selected as required). The resulting expression vector for the BoNT / A protein precursor was then transformed into competent cells to create engineered cells (engineered bacteria). Protein expression in these engineered cells yielded the BoNT / A single-chain protein, a process that follows standard procedures. In subsequent experiments, Hind III and Nde I restriction enzyme sites were inserted at both ends of the BoNT / A single-chain protein nucleotide fragment. Then, using conventional restriction enzyme digestion and ligation methods, the BoNT / A single-chain protein nucleotide fragment was integrated into the commercially available pET-26b empty expression vector via the Hind III and Nde I restriction sites, thus obtaining the expression vector for the BoNT / A single-chain protein (pET-26b-BoNT / A). The plasmid map and ligation process diagram are shown in Figure 4. The expression vector was transformed into *E. coli*, and engineered bacteria expressing recombinant BoNT / A protein (BoNT / A protein precursor) were obtained through routine transformation and screening. The engineered bacteria specifically selected for subsequent experimental studies was *E. coli* BL21(DE3). *E. coli* strains include, but are not limited to, BL21(DE3), BL21(DE3)plysS, JM109(DE3), and Rosetta(DE3); BL21(DE3) is more preferred. The above methods are all conventional molecular cloning techniques (recombinant technology) in the prior art, and can be found in reference books such as *Molecular Cloning: A Laboratory Manual*, and will not be elaborated further here.

[0133] Example 2: Recombinant BoNT / A Expression and Protein Purification

[0134] The recombinant BoNT / A engineered bacteria were inoculated into shake flasks containing LB medium and cultured until the OD600 reached 1.6-2.5. Then, the culture was transferred to a 5L fermenter with an initial culture volume of 2L, a culture temperature of 37℃, and a stirring speed of 300-800rpm. Induction was initiated when the OD600 value reached 20-25 using isopropyl-β-D-thiogalactoside (IPTG) at a concentration of 0.5mM for 3-20 hours. The bacterial growth in the fermentation broth was examined under a microscope. After 3-20 hours of induction, the fermentation broth was collected and centrifuged at 4℃ and 7000rpm for 10 minutes to collect the bacterial cells.

[0135] The obtained bacterial cells were sequentially subjected to lysis, Ni column chromatography, cation exchange chromatography, enzyme digestion, and cation exchange chromatography. Except for the enzyme digestion step which used a specific Lys-C enzyme, the above processes were standard procedures for extracting and purifying proteins from bacterial cells using existing technologies. The specific processing steps are described below:

[0136] (1) Collection of bacterial cells: The fermentation liquid was centrifuged at 7500 r / min for 10 min in a refrigerated centrifuge to collect the bacterial cells. The centrifuged bacterial cells were then frozen at -20℃.

[0137] (2) Bacterial dispersion and lysis: Bacterial cells were added to lysis buffer (PBS) at a ratio of 100 g / L and stirred to suspend. After the absence of large particles, the cells were sonicated and then centrifuged at 7500 r / min, 10 min, and 4 °C in a refrigerated centrifuge. The supernatant was collected.

[0138] (3) Ni column purification:

[0139] The reagents used include: equilibration buffer A: 20 mM sodium dihydrogen phosphate, pH 7.0; and elution buffer B: 20 ​​mM sodium dihydrogen phosphate, 0.3 M imidazole, pH 7.0.

[0140] The supernatant collected by centrifugation was loaded onto a pre-equilibrated Ni column (Monmix MC60-NTA Ni), reequilibrated with equilibration buffer A, and then eluted using elution buffer B. The target peak containing the BoNT / A single-chain protein was collected to obtain the Ni column purified product. Ni column purification mainly separates the histidine-tagged fusion protein (BoNT / A single-chain protein) from other proteins.

[0141] (4) Cation exchange chromatography:

[0142] The reagents used include: Equilibrium buffer A: 20 mM sodium dihydrogen phosphate, pH 6.5; Elution buffer B: 20 ​​mM sodium dihydrogen phosphate, 0.5 M sodium chloride, pH 6.5.

[0143] The Ni-column purified product obtained from Ni-column recovery was loaded onto a pre-equilibrated 30S column (BestPoly 30S). After reequilibration with equilibration buffer A, routine elution was performed using elution buffer B, and the target peak containing the BoNT / A single-chain protein was collected to obtain the first ion-exchange purified product. The target protein and other contaminating proteins were further separated by cation exchange chromatography.

[0144] (5) Enzyme digestion:

[0145] Adjust the pH of the cation exchange chromatography sample to 8.0-8.5, and add 0.05-1 mg of Lys-C enzyme at a ratio of 1 g BoNT / A single-chain protein for enzymatic digestion (i.e., the mass ratio of Lys-C enzyme to BoNT / A single-chain protein is controlled at 1:1000-1:20000). Digest at 20-25℃ for 2-24 h.

[0146] Preferably, the enzyme digestion conditions are: pH 8.0, the ratio of BoNT / A single-chain protein to Lys-C enzyme is 1g:1mg, the digestion time is 4h, and the digestion temperature is 25℃.

[0147] The BoNT / A single-chain protein is cleaved into heavy and light chains by enzymatic digestion. The sequences of both heavy and light chains are identical to those of the light and heavy chains in the native BoNT / A double-chain protein, with the linker peptide removed by the digestion. The heavy and light chains then spontaneously reassemble to form the correctly conformated BoNT / A protein. The sequence design and digestion method used in this protocol for the BoNT / A single-chain protein are as follows: "...TSK↓TKSLDKGYNK..." and "...GGGGSPK↓ALNDL". Digestion completely removes the linker region "GGGGSGGGGSGGGSHHHHHHGGGGSGGGGSGGGSPK" and the C-terminal decapeptide "TKSLDKGYNK" of the first functional region (light chain). The remaining light and heavy chain sequences are identical to those of the native BoNT / A protein. In this protocol, digestion occurs at two specific lysine residues in the BoNT / A single-chain protein. Although the BoNT / A single-chain protein also has other lysine sites, it forms a specific structure (an incorrect structure relative to the native protein) at the two lysine sites mentioned above. This structure is easily recognized and cleaved by the Lys-C enzyme, while cleavage does not occur at other lysine sites, ensuring the formation of the correct heavy and light chain sequences. Therefore, the upstream design of the BoNT / A single-chain protein in this scheme, unlike existing technologies, guarantees the acquisition of the native BoNT / A protein structure.

[0148] (6) Cation exchange chromatography:

[0149] The reagents used include: Equilibrium buffer A: 20 mM sodium dihydrogen phosphate, pH 6.5; Elution buffer B: 20 ​​mM sodium dihydrogen phosphate, 0.5 M sodium chloride, pH 6.5.

[0150] After dilution, the enzyme-digested sample was adjusted to pH 6.5 and loaded onto a pre-equilibrated 30S column (BestPoly 30S). After reequilibration with equilibration buffer A, routine elution was performed using elution buffer B, collecting the target peak containing BoNT / A protein. This step involved cation exchange chromatography to further remove irrelevant proteins. The collected product was the final BoNT / A product, i.e., the stock solution. The eluted sample was subjected to SDS-PAGE electrophoresis, as shown in Figure 5. In Figure 5A, from left to right, lane 1 represents the unreduced single-chain protein before enzyme digestion; lane 2 represents the reduced single-chain protein before enzyme digestion; lane 3 is the marker; lane 4 represents the unreduced stock solution (BoNT / A protein); and lane 5 represents the reduced stock solution (the reduced BoNT / A protein separated into heavy and light chains). Figure 5B shows the SDS-PAGE electrophoresis results of the final stock solution. From left to right, lane 4 is the unreduced stock solution (BoNT / A protein); lane 2 is the marker; and lane 3 is the reduced stock solution (the reduced solution is separated into heavy and light chain BoNT / A protein). The Lys-C residue in the stock solution prepared in this example was detected using ELISA, and the residue level was less than 0.1 ng / mg (Lys-C enzyme mass / botulinum toxin protein mass).

[0151] Example 3: Characterization of recombinant BoNT / A protein

[0152] (1) Complete molecular weight analysis

[0153] Mass spectrometry conditions: The test sample was analyzed by mass spectrometry using a Xevo G2-XS QTof mass spectrometer for 15 minutes.

[0154] Detection method: positive ion, precursor ion scanning range: 500-4000 m / z.

[0155] The test sample was separated using an ultra-high performance liquid chromatography (UHPLC) system. Solution A was an aqueous solution containing 0.1% formic acid, and solution B was an acetonitrile solution containing 0.1% formic acid. The column was equilibrated with solution A. The test sample was loaded via an autosampler and then separated by the column at a flow rate of 0.3 mL / min, a detection wavelength of 280 nm, and a column temperature of 80 ℃.

[0156] The molecular weight of the intact protein was determined to be 148,172.9 Da using liquid chromatography-mass spectrometry (LC-MS) and software analysis. The UV-coded plot of the intact molecular weight of the test sample is shown in Figure 6; the deconvolutioned bar chart of the intact molecular weight of the test sample is shown in Figure 7.

[0157] (2) Reduced molecular weight analysis

[0158] Add an appropriate amount of the test sample to a 6 M denaturing buffer solution of guanidine hydrochloride, add DTT to a final concentration of 100 mM, and incubate at 100 °C for 5 minutes. Load the test sample using an autosampler, then separate it using a chromatographic column at a flow rate of 0.3 mL / min, a detection wavelength of 280 nm, and a column temperature of 80 °C.

[0159] After liquid chromatography-mass spectrometry (LC-MS) and software analysis, the molecular weight of the reduced light chain protein was determined to be 50024.4 Da, and the molecular weight of the heavy chain protein was 98151.6 Da. The UV-coded molecular weight chart of the reduced sample is shown in Figure 8; the deconvolutioned complete molecular weight histogram of the reduced light chain is shown in Figure 9; and the deconvolutioned complete molecular weight histogram of the reduced heavy chain is shown in Figure 10.

[0160] Chinese patent CN114957482B describes the natural BoNT / A protein: The natural BoNT / A light chain possesses a natural loop region at its C-terminus. Under natural conditions, this loop is cleaved by an unidentified clostridium endopeptidase at the latest during cell lysis when toxins are released from Clostridium. For BoNT / A, the loop sequence on its light chain can be cleaved to yield a decapeptide (i.e., the aforementioned TKSLDKGYNK). The cleaved light and heavy chains are connected by a disulfide bond at their respective N-terminus and C-terminus, with a cysteine ​​residue at each.

[0161] The light chain sequence of the naturally occurring, active BoNT / A product (a dimer formed by the heavy and light chains) is actually SEQ ID NO.1 with the C-terminal TKSLDKGYNK sequence removed and the N-terminal M (E. coli possesses its own methionine aminopeptidase; after E. coli expresses the protein, the initial M is automatically removed without further processing). Its theoretical molecular weight is approximately 50024 Da (i.e., SEQ ID NO.11, see below). The theoretical molecular weight of the natural BoNT / A heavy chain is approximately 98151 Da (i.e., SEQ ID NO.3).

[0162] Among them, naturally extracted type A botulinum toxin refers to the commercially available products Botox and Xeomin.

[0163] For details on Botox, please see https: / / www.genome.jp / entry / D08957;

[0164] For details on Xeomin, please see https: / / www.pmda.go.jp / drugs / 2020 / P20200703001 / .

[0165] The heavy chain sequence of naturally extracted type A botulinum toxin (a dimer of light and heavy chains formed after cleavage of a single-chain protein) is shown in SEQ ID NO.3, and the light chain sequence is shown in SEQ ID NO.11.

[0166] PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTS IVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVT LAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSG KFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK.

[0167] This protocol involves plasmid construction, engineered bacterial expression, enzymatic digestion, and purification to obtain a BoNT / A light chain with a molecular weight identical to that of the naturally extracted BoNT / A double-stranded light chain. Similarly, the obtained BoNT / A heavy chain has a molecular weight substantially identical to that of the naturally extracted BoNT / A double-stranded heavy chain. Through single-chain protein sequence design and enzymatic digestion, all linker peptides located between the heavy and light chains are removed, resulting in BoNT / A light and heavy chains with structures identical to those extracted from natural sources.

[0168] (3) Disulfide bond analysis and free thiol group analysis

[0169] After denaturation and NEM alkylation, the sample was replaced with 20 mM PB buffer. A portion was added to Lys-C and the reaction was carried out at 37 °C for 18 h. The reaction was terminated by acidification with 7 M Gdn-HCl, and a portion of the digest was taken and reduced with 1 M TCEP. The digest products of the test sample were separated using a liquid chromatography system.

[0170] The enzymatic hydrolysis products of the test sample were desalted and separated by high performance liquid chromatography (HPLC) and then analyzed by high resolution mass spectrometry (HMS). Analysis time: 120 min; detection mode: positive ion (MSE); precursor ion scan range: 300-2000 m / z.

[0171] After non-reducing enzymatic hydrolysis and liquid chromatography-mass spectrometry analysis, two pairs of disulfide bonds consistent with the theoretical disulfide bond pairing pattern were identified: C429-C443 and C1224-C1269. From the perspective of the finished BoNT / A product (where the light and heavy chains have been cleaved and bonded by disulfide bonds), the 429th amino acid of its light chain is cysteine, and the 6th amino acid of its heavy chain is also cysteine, forming a disulfide bond between them (LC429 and HC6). Similarly, the 787th and 832nd amino acids of its heavy chain are both cysteine, forming disulfide bonds between them (HC787 and HC832). The disulfide bond formation is consistent with that of naturally extracted BONT / A.

[0172] The test sample was lyophilized and concentrated, diluted with 20 mM PB solution, denatured, and alkylated with NEM, then replaced with 20 mM PB buffer. A portion was added to Lys-C and enzymatically digested at 37 °C for 18 h. The reaction was terminated by acidification with 7 M Gdn-HCl. A portion of the digested product was reduced with 1 M TCEP. The digested product was desalted and separated by high-performance liquid chromatography (HPLC) and then analyzed by high-resolution mass spectrometry (HS-MS). Analysis time: 120 min; detection mode: positive ion (MSE); precursor ion scan range: 300-2000 m / z. Mass spectrometry data were retrieved using UNIFI (Waters) software for theoretical free thiol peptide data retrieval and post-reduction validation analysis.

[0173] The identification results of the alkylated modified peptides of the test samples are detailed in Table 1, and the proportion of free thiol groups of the test samples is detailed in Table 2. The primary and secondary mass spectrometry identification spectra of the test sample peptide (1:K12&:NEM (2)

[0637] +H⁺) are detailed in Figure 11, the primary and secondary mass spectrometry identification spectra of the test sample peptide (2:K25&:NEM [5]+H⁺) are detailed in Figure 12, the primary and secondary mass spectrometry identification spectra of the test sample peptide (2:K38&:NEM

[0016] +H⁺) are detailed in Figure 13, and the primary and secondary mass spectrometry identification spectra of the test sample peptide (2:K44&:NEM [4]+H⁺) are detailed in Figure 14.

[0174] Table 1: Identification results of alkylated modified peptides of the test samples (including: observed retention time (RT), expected mass, observed mass, mass error, etc.)

[0175] Name Peptide Sequence Observation Retention Time (min) Expected Quality (Da) Observed Quality (Da) Quality Deviation (ppm) 1:K12&:NEM (2)

[0637] +H⁺VIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECK87.594456.20514456.20530.12:K25&:NEM [5]+H⁺FLNQCSVSYLMNSMIPYGVK84.602419.14952419.1484-0.52:K38&:NEM

[0016] +H⁺YFNSISLNNEYTIINCMENNSGWK79.612979.32882979.3278-0.32:K44&:NEM [4]+H⁺LDGCRDTHRYIWIK45.061900.94871900.9437-2.6

[0176] Table 2: Proportion of Free Thiol Groups in Test Samples

[0177] Name Free thiol ratio %1:K12&:NEM (2)

[0637] +H⁺1002:K25&:NEM [5]+H⁺1002:K38&:NEM

[0016] +H⁺1002:K44&:NEM [4]+H⁺100

[0178] Analysis revealed that the test sample was subjected to alkylation of free thiol groups by NEM followed by enzymatic cleavage and reduction. Liquid chromatography-mass spectrometry analysis showed that cysteine ​​was mainly present in a free state, with contents of C133:100%, C164:100%, C780:100%, C956:100%, and C1049:100%.

[0179] (4) N-terminal sequence analysis

[0180] Electrophoresis: Take a sufficient amount of the test sample, add it to the reducing sample buffer, mix well and boil. Load the test sample into two wells and add the pre-stained marker. Stack the gel at 100 V for 15 min and the separating gel at 180 V for 30 min.

[0181] Transfer: Prepare a PVDF membrane, cut to the same size as the gel, and wet it evenly in 100% methanol. Place the gel on thick filter paper also soaked in transfer buffer, then place the PVDF membrane on top of the gel, cover with another wetted thick filter paper, expel air to fill with liquid, and finally clamp them together in the electrotransfer tank. Place the transfer clamp in the transfer tank with the gel side facing the negative terminal, add transfer buffer, turn on the external circulation cooling to 10 °C, and turn on the magnetic stirrer under the electrotransfer tank, adjusting the stir bar to low speed. Turn on the power and perform wet transfer at 250 mA for 90 min. After the power is applied, remove the transfer clamp, remove the PVDF membrane, stain it with dye solution, and then rinse with purified water until the bands are clear. Scan with EPSON and upload the image, then cut out the desired bands and dry the test sample.

[0182] On-machine testing:

[0183] Place the cut PVDF membrane into the reactor, assemble the reactor, and place it in the fixed position on the instrument. Use the PPSQ Analysis software to set the sample name, number, number of test cycles, and method file, and start the N-terminal test.

[0184] Data and graph processing:

[0185] The raw data and spectra generated by PPSQ are identified and peaks are marked by PPSQ DataProcessing software, which then exports the corresponding spectra.

[0186] Based on the above analysis, the N-terminal sequencing results of the test sample are as follows:

[0187] Heavy chain: NH₂-Ala-Leu-Asn-Asp-Leu-X-Ile-Lys-Val-Asn-Asn-Trp-Asp-Leu-Phe.

[0188] (ALNDLXIKVNNWDLF, Cys corresponds to X, consistent with the theoretical sequence)

[0189] Light chain: NH₂-Pro-Phe-Val-Asn-Lys-Gln-Phe-Asn-Tyr-Lys-Asp-Pro-Val-Asn-Gly.

[0190] (PFVNKQFNYKDPVNG)

[0191] Therefore, it is evident that the N-terminal sequences of both the heavy and light chains of the BoNT / A prepared using this method are identical to those of the naturally extracted BoNT / A, without any inserted or missing amino acid residues. Using this technique, the N-terminal sequence of the heavy chain is completely identical to SEQ ID NO.3, and the N-terminal sequence of the light chain is completely identical to SEQ ID NO.5, with no undesirable intermediate products. Furthermore, the enzymatic digestion method used in this method will not introduce any other amino acid residues from non-natural heavy chain components into the N-terminus of the heavy chain.

[0192] (5) C-terminal analysis

[0193] Enzymatic hydrolysis of the test sample: The lyophilized and concentrated test sample was diluted with 20 mM PB solution, and an equal volume of 8 M UA was added. The sample was denatured at 37 ℃ for 1 h. Lys-C was added, and the enzymatic hydrolysis reaction was carried out at 37 ℃ for 18 h. The reaction was terminated by acidification with 7 M Gdn-HCl, and 1 M TCEP was added for reduction.

[0194] Liquid chromatography-mass spectrometry (LC-MS): Enzymatically digested samples were separated using an ACQUITY UPLC I-Class system.

[0195] After separation, high-resolution mass spectrometry (HPLC) was used for detection and scanning mass spectrometry analysis. Analysis time: 120 min; detection mode: positive ion (MSE); precursor ion scan range: 300-2000 m / z.

[0196] Mass spectrometry data processing: Mass spectrometry data were processed using the UNIFI control program (1.9.4, Waters). The theoretical sequence of the test sample was selected as the database, and then a database matching search was performed. Peptide information obtained from Lys-C digestion of the test sample was analyzed by liquid chromatography-mass spectrometry (LC-MS) and the data was then used to search the theoretical sequence database and perform matching analysis to identify C-terminal peptide sequences of both the light and heavy chains.

[0197] The BPI spectrum of Lys-C enzymatic digestion products is shown in Figure 15. The component plot mass spectrum of the Lys-C cleavage light chain C-terminal peptide of the test sample is shown in Figure 16. The primary identification mass spectrum of the Lys-C cleavage light chain C-terminal peptide of the test sample is shown in Figure 17. The secondary identification mass spectrum of the Lys-C cleavage light chain C-terminal peptide of the test sample is shown in Figure 18. The component plot mass spectrum of the Lys-C cleavage heavy chain C-terminal peptide of the test sample is shown in Figure 19. The primary identification mass spectrum of the Lys-C cleavage heavy chain C-terminal peptide of the test sample is shown in Figure 20. The secondary identification mass spectrum of the Lys-C cleavage heavy chain C-terminal peptide of the test sample is shown in Figure 21.

[0198] Based on the above experimental results, the light chain C-terminal sequence of the test sample LPJT-099 stock solution (batch number: 20240601) was identified by mass spectrometry analysis as: LLCVRGIITSK;

[0199] The heavy chain C-terminal sequence is: LVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL.

[0200] Consistent with the theoretical C-terminal sequences of the heavy and light chains, the BoNT / A protein prepared by this method has the same sequence as the natural BoNT / A protein (molecular weight, C-terminus, and N-terminus are all the same).

[0201] Based on the above molecular weight analysis, amino acid terminology analysis, and disulfide bond analysis, it is evident that the BoNT / A synthesized using this method is identical to the natural BoNT / A in terms of amino acid sequence (primary structure), light and heavy chain composition, and disulfide bond formation. Therefore, the preparation method described in this technique can yield botulinum toxin BoNT / A with a double-stranded morphology consistent with the structure extracted from natural endotoxin.

[0202] (6) Circular dichroism analysis

[0203] After soaking the cuvettes in 2M HNO3 overnight and rinsing them thoroughly with deionized water, they were air-dried. Background and blank buffer were scanned sequentially. Then, an appropriate amount of the test sample was added to the cuvettes for near-UV (250-340 nm) and far-UV (190-260 nm) scanning. The near-UV CD spectrum of the test sample is shown in Figure 22, and the far-UV CD spectrum is shown in Figure 23.

[0204] The scanned spectra were processed using Pro-Data Viewer software with average and smoothing effects, and the smoothing times were set to 3. The ratio of peak to trough CD values ​​of the standard sample was calculated, and the effective ratio range was 2.08 ± 0.06. Secondary structure prediction of the spectra was performed using CDNN software.

[0205] The secondary structure of the test samples was fitted and calculated using CDNN software, 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 sample are shown in Table 3.

[0206] Table 3: Statistical Table of Secondary Structure Prediction for Each Test Sample

[0207] HelixAntiparallelParallelBeta-TurnRandom Coil19.4%28.7%6.4%18.1%29.2%

[0208] Example 4: Activity assay of recombinant BONT / A protein (stock solution toxicity)

[0209] (1) Experimental design and grouping of drugs:

[0210] Sixty male Kunming mice (SPF grade) aged 26-30 days were selected from those with similar body weights after acclimatization and randomly divided into 6 groups of 10 mice each. Samples were used to prepare the test sample. The samples were diluted to the same concentration. The toxic reactions of the mice and the mortality of each group were closely observed for 4 consecutive days after administration. The sample is the sample prepared in Example 2. The eluent obtained after cation exchange chromatography in "(6) Cation Exchange Chromatography" is called the stock solution.

[0211] (2) Experimental procedure:

[0212] Before the experiment, the mice were weighed and distributed equally among the groups to ensure that there was no statistically significant difference in the average weight of each group. There were 6 groups, with 10 male mice in each group. The drug was administered via intraperitoneal injection; one person drew the test solution, while another person verified and completed the administration to the animals. The time of administration was recorded after each injection. The grouping and dosage information for the test product, botulinum toxin type A, is shown in Table 4.

[0213] Under the experimental conditions, the LD50 of the recombinant botulinum toxin type A stock solution was calculated using SPSS. 50 The concentration was 3.917 pg / animal, which translates to a toxicity of 2.55 × 10⁻⁶ pg / animal. 8 LD 50 / mg, which is comparable to the toxicity of wild-type BONT / A in existing technologies.

[0214] Table 4: Results of toxicity test of BONT / A protein obtained in Example 2

[0215] Test sample solution content (pg / mL) Dosage (pg / animal) Total number of animals (animals) Total number of deaths (animals) 6.003 100 8.004 106 10.60 5.3 1010 14.1 7.05 1010 18.80 9.4 1010 25.00 12.5 1010

[0216] Example 5: DAS scoring

[0217] Forty healthy, sexually mature female SD rats (SPF grade), weighing approximately 180-200g, were randomly divided into four groups (n=10 / group) after acclimatization: solvent control group, low-dose group, medium-dose group, and high-dose group. Samples were taken and administered via a single unilateral intramuscular injection. Post-administration, the rats' hind toe abduction (DAS) was closely observed and scored according to a scoring scale (see Figure 24 for scoring criteria; reference: Ron S Broide, The rat Digit Abduction Score (DAS) assay: a physiological model for assessing botulinum neurotoxin-induced skeletal muscle paralysis, Toxicon. 2013 Sep:71:18-24. doi: 10.1016 / j.toxicon.2013.05.004. Epub 2013 May 23.).

[0218] Before the experiment, rats were evenly distributed into groups based on their body weight to ensure no statistically significant difference in average body weight among the groups. Samples were diluted to the appropriate concentrations. The experiment consisted of four groups: a solvent control group, a low-dose group (5 U / kg), a medium-dose group (10 U / kg), and a high-dose group (20 U / kg). A single dose was administered via multiple injections into the gastrocnemius muscle of one hind limb of the animal; the injection volume at each point did not exceed 0.20 mL. Animals were scored using a scoring system before and after the drug administration on days D1, D2, D3, D4, D5, D7, D9, D11, D15, D19, D23, D28, D35, and D42. The experimental dosage and grouping information are shown in Table 5 below.

[0219] Table 5: Experimental grouping and drug administration arrangements

[0220] Group Dosage (U / kg) Administration Concentration (U / mL) Administration Volume (mL / kg) Number of Animals Solvent Control Group -- 0.210 Low-dose group 525 0.210 Medium-dose group 1050 0.210 High-dose group 20100 0.210

[0221] The experimental results are shown in Table 6. The results show that the application of BONT / A prepared in this scheme can effectively block neuromuscular transmission, inhibit muscle contraction, and achieve denervation.

[0222] Table 6: DAS score results of BONT / A protein obtained in Example 2

[0223] Dosage Pre-drug D1 D2 D3 D4 D5 D7 D9 D11 D15 D19 D23 D28 D35 D42 Solvent Control Group 0000000000000005 U / kg 00000 0.3 0.4 0.5 0.6 0.4 0.3 0.2 0.2 0.1 0.1 0.1 10 U / kg 000.2 0.7 1.3 1.6 1.8 1.8 1.5 1.4 1.1 0.8 0.7 0.4 0.3 20 U / kg 00.3 1.4 1.9 2.5 3.3 3.7 3.8 3.1 2.8 2.2 2.1 1.8 0.8 0.5

[0224] Comparative Example 1

[0225] In the prior art, endotoxins with natural structures are mainly obtained through two methods: extraction from Clostridium botulinum; and expression of heavy and light chains in different engineered bacteria, followed by assembly.

[0226] Existing technologies also attempt to integrate the heavy and light chains of botulinum toxin to form a fusion protein, express it in the same engineered bacteria, and then obtain botulinum toxin through enzymatic digestion and other operations. For example, Chinese patent CN114957482B (A modified neurotoxin single-chain polypeptide and its use) uses a technology that sequentially links a tag protein, a light chain, and a heavy chain to form a fusion protein, with enzyme cleavage sites set between the light and heavy chains and between the tag protein and the light chain. Specifically, the botulinum toxin precursor sequence in this patent technology is as follows: tag protein, first protease cleavage site LEVLFQGPL, linking short peptide GS, light chain of the first functional amino acid structural region BoNT / A (C-terminus: ...CVRGIITS, amino acid residues in the loop region of the C-terminus were removed in advance during sequence design), second protease cleavage site LEVLFQGP, heavy chain of the second functional amino acid structural region BONT / A (N-terminus: ALNDLCIK...). The recognition sequence LEVLFQGP of the Tobacco Etch Virus (TEV) protease is followed by cleavage (LEVLFQ↓GP), and then recombination forms a dimer of light and heavy chains (double-chain botulinum toxin). However, this method cannot obtain botulinum toxin with a natural structure. For example, the light chain sequence is not a natural sequence; it still contains residues with cleavage sites at its C-terminus and N-terminus, as well as a short GS peptide. Similarly, the heavy chain is not a natural sequence; it also retains amino acid residues with cleavage sites at its N-terminus. Because the botulinum toxin obtained by this patented technology contains amino acid residues that are not naturally occurring in botulinum toxin, it is not a botulinum toxin with a natural structure and differs from the proposed technical solution. Sequence alignment can be seen in Figures 1-3.

[0227] Chinese patent CN118006523A (Recombinant Genetically Engineered Bacteria for Type A Botulinum Toxin and Its Preparation and Application) also attempted to produce recombinant type A botulinum toxin. The specific method involves integrating a recombinant single-chain polypeptide of botulinum toxin using the pET-28a vector, followed by protein expression, cleavage, and purification to ultimately form type A botulinum toxin. The recombinant single-chain polypeptide includes gene fragment 1 expressing a GST tag, gene fragment 2 expressing a thrombin recognition site, and gene fragment 3 expressing type A botulinum toxin BoNT / A. The BoNT / A light chain-heavy chain junction region includes the amino acid sequence of GST, another thrombin recognition site. The thrombin recognition site is LVPRGS, and the cleavage site is LVPR↓GS. Similarly, the N-terminus of the heavy chain of the BONT / A formed by cleavage 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. Sequence alignment results can be seen in Figures 1-3.

[0228] The unique design of the BONT / A precursor (single-chain polypeptide) in this solution ensures that, after enzymatic digestion, the heavy and light chains of BONT / A with a structure identical to that of naturally extracted BONT / A are obtained, resulting in a product that is structurally identical to naturally extracted BONT / A. Since naturally extracted BONT / A is widely used, its safety and efficacy can be effectively guaranteed. This novel BONT / A preparation method improves the efficiency of the preparation process while ensuring complete structural consistency between the product and the naturally extracted one. It offers significant advantages over existing technologies that prepare BONT / A using single-chain polypeptides of recombinant proteins.

[0229] Comparative Example 2

[0230] Existing technologies typically involve expressing the light and heavy chains separately, then combining them to form BoNT / A. For example, Chinese patent CN115894641B (Construction of Type A Botulinum Toxin Mutant and its Genetically Engineered Bacteria) expresses the light and heavy chains separately and mutates the cysteine ​​residues of both chains. However, expressing the light and heavy chains separately increases the complexity of the process. Furthermore, the mutated light and heavy chains in this approach result in a product with a significantly different structure from the naturally extracted product; therefore, its safety and efficacy require further investigation. Compared to the product with a structure identical to naturally extracted BoNT / A, achieving clinical application of this product presents greater challenges and a longer timeframe.

[0231] Comparative Example 3: Study on the introduction location of the restriction enzyme site

[0232] This comparative study explored the setting of restriction enzyme cleavage sites extensively. The first attempt was to set the cleavage site at the N-terminus of a single-chain protein, the amino acid sequence of which is shown below (SEQ ID NO.9):

[0233]

[0234] In the above sequence, a histidine tag and a linker sequence (double-strikethrough) were added to the N-terminus of the light chain. The light and heavy chains were directly linked by a decapeptide, unlike the previous method which used a linker peptide containing a histidine tag. A restriction enzyme site “AEAEAPK↓PFVNKQFNY” was set at the N-terminus of the light chain, and cleavage was planned to occur there, resulting in a light chain N-terminus identical to 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 SEQ ID NO.9, and the restriction enzyme digestion method was performed exactly as preferred in Example 2. The N-terminus / C-terminus of the obtained botulinum toxin BoNT / A were sequenced. The light chain N-terminus was SDKII (no cleavage occurred at the designed restriction enzyme site), and the heavy chain N-terminus was ALNDL (identical to the natural sequence). The C-terminus were identical to the natural sequence. Sequencing results showed that the N-terminus of the light chain did not form the expected cleavage pattern, and the structure of the obtained botulinum toxin was inconsistent with that of natural botulinum toxin. Therefore, adding a tag to the N-terminus of the single-chain protein is not suitable.

[0235] Then, an attempt was made to place the restriction enzyme site at the C-terminus of the single-chain protein, the amino acid sequence of which is shown below (SEQ ID NO.10):

[0236]

[0237] In the above sequence, a histidine tag and a linker sequence (double-strikethrough) were added to the C-terminus of the heavy chain. The light and heavy chains were directly linked by a decapeptide (in the natural form), unlike the method described in this study which uses a linker peptide containing a histidine tag between them. 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 digestion method was performed exactly as preferred in Example 2. The molecular weights of the light and heavy chains of the obtained botulinum toxin BoNT / A were determined (reduced molecular weight). The molecular weight of the light chain was 50025.8, while the molecular weight of the heavy chain varied: 98280.60, 95564.5, and 95039.0. The results showed that the light chain was consistent with the theory, while the heavy chain showed incomplete or incorrect digestion. The molecular weight determination results indicated that, according to the above single-chain protein sequence design, the C-terminus of the heavy chain was difficult to form the enzyme digestion method expected in the design sequence, and the structure of the obtained botulinum toxin was inconsistent with that of natural botulinum toxin. Therefore, adding a tag to the C-terminus of a single-chain protein is not appropriate.

[0238] This technical solution designs the histidine tag between the light and heavy chains. The addition of the histidine tag facilitates protein purification. Through enzymatic cleavage with Lys-C enzyme, 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, unexpected technical effects have been achieved.

[0239] Comparative Example 4: Study on Enzyme Digestion Methods

[0240] The single-chain protein sequence of SEQ ID NO.10 in Comparative Example 3 could not be correctly cleaved at the C-terminus of the heavy chain using the enzyme digestion method of Example 2, but it could correctly cleave the C-terminus of the light chain and the N-terminus of the heavy chain. The linkage between the heavy and light chains 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 linked to the heavy chain through the aforementioned decapeptide).

[0241] More specifically, the enzymatic digestion method for single-chain proteins was as follows: pH 8.0, a ratio of BoNT / A single-chain protein to Lys-C enzyme of 1g:1mg (enzyme to single-chain protein ratio of 1:1000), digestion time of 4 hours, and digestion temperature of 25℃. Under these conditions, the C-terminus of the light chain and the N-terminus of the heavy chain could be correctly cleaved (the obtained product was consistent with the natural sequence). However, the amount of Lys-C enzyme used in this digestion method was still relatively large, which did not take advantage of saving raw materials or reducing impurities in the final product. An attempt was made to reduce the amount of enzyme and observe the digestion results. The digestion method for the single-chain protein of SEQ ID NO.10 was modified to: pH 8.0, 1g of BoNT / A single-chain protein was added to 0.05mg of Lys-C enzyme for digestion (enzyme to single-chain protein ratio of 1:20000), and digestion was carried out at 20℃ for 16 hours. The remaining procedures were the same as in Example 2, except for the enzyme digestion conditions. The digestion product was prepared as described in Example 2. Analysis of the proteins in the digest revealed that a large number of heavy and light chains remained uncleaved. Specifically, after reducing the proteins in the digest and performing electrophoresis, in addition to the light and heavy chain bands, a band with a relatively large molecular weight (approximately the sum of the molecular weights of the light and heavy chains) appeared, indicating that the light and heavy chains were not fully cleaved under the given enzyme digestion conditions. Furthermore, the reduced molecular weights were analyzed. Besides the correct light chain molecular weight (approximately 50024 Da) and heavy chain molecular weight (approximately 98151 Da), the reduced molecular weights of both the light and heavy chains exhibited various forms, either slightly larger or slightly smaller than the correct molecular weight. This indicates that the light and heavy chains are connected in a natural manner. Achieving correct cleavage of the C-terminus of the light chain and the N-terminus of the heavy chain to form a sequence conforming to the natural structure requires specific requirements for the enzyme digestion method (especially the amount of Lys-C enzyme).

[0242] The enzyme digestion method for the single-chain protein sequence (SEQ ID NO. 7) of this scheme was studied. Specifically, the digestion method was as follows: 1g of BoNT / A single-chain protein was digested with 0.05mg of Lys-C enzyme at pH 8.0 (enzyme to single-chain protein ratio of 1:20000) at 20℃ for 16h. The remaining procedures were the same as in Example 2, only the digestion conditions were changed. The digested product was prepared into a stock solution according to the method in Example 2. The reduced molecular weight was determined, and the results were consistent with the theory: light chain: approximately 50024.00, heavy chain: approximately 98151.75, with no other molecular weight variations observed. There were no instances of incomplete cleavage between the light and heavy chains, nor were there any errors in the cleavage sites at the C-terminus of the light chain or the N-terminus of the heavy chain. The sequences of both the light and heavy chains of the obtained botulinum toxin product were consistent with the natural sequence, and the cleavage effect was consistent with that of increasing the amount of Lys-C enzyme in Example 2, resulting in a botulinum toxin product with a structure identical to the natural structure. The Lys-C residue in the stock solution prepared by this method was detected by ELISA, and the residue level was less than 0.1 ng / mg (Lys-C enzyme mass / botulinum toxin protein mass).

[0243] Based on the experimental results above, it is evident that this method incorporates a histidine-tagged linker peptide between the light and heavy chains. The presence of this histidine tag significantly enhances the efficiency of subsequent purification processes. The linker peptide ensures correct cleavage between the light and heavy chains, with the C-terminus of the light chain and the N-terminus of the heavy chain perfectly matching their natural structures. Furthermore, the linker peptide significantly reduces the amount of enzyme required for digestion. Even with an enzyme-to-single-chain protein ratio of 1:20000, the C-terminus of the light chain and the N-terminus of the heavy chain can still achieve correct cleavage, preventing incomplete digestion or incorrect cleavage sites. Therefore, the upstream design of this botulinum toxin product differs from conventional methods. By using a histidine-tagged linker peptide between the light and heavy chains, it ensures that the final product sequence is completely identical to natural botulinum toxin. It also significantly reduces enzyme usage during single-chain polypeptide digestion, thereby lowering reagent costs, reducing product impurities, and simplifying subsequent purification processes.

[0244] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described 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 are, in order: BoNT / A-LC—linker—BoNT / A-HC, where, (1) BoNT / A-LC is a type A botulinum toxin light chain or a modified type A botulinum toxin light chain; (2) The linker is the linker region; the linker region includes the linker peptide, the tag protein polypeptide, the linker peptide and the protease recognition site in sequence; (3) BoNT / A-HC is a type A botulinum toxin heavy chain.

2. The single-chain protein of recombinant botulinum toxin type A according to claim 1, characterized in that: The amino acid sequence of the type A botulinum toxin light chain is shown in SEQ ID NO.1; the amino acid sequence of the modified type A botulinum toxin light chain is shown in SEQ ID NO.2; and the amino acid sequence of the type A botulinum toxin 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 region is as follows: (Connecting peptide) a —(Tag protein polypeptide) b — (Connecting peptide) c —Protein recognition site; Where 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 linker peptide is GGGGS, GGGS, GGS, GGGQ or GEQP; The tagged protein polypeptide is selected from histidine tags; preferably, the sequence of the histidine tag is selected from HHHHHH or HQHQHQ.

5. A 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; where 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 recombinant botulinum toxin type A according to any one of claims 1-5, characterized in that: Recombinant botulinum toxin type A was obtained by digesting the single-chain protein of recombinant botulinum toxin type A with Lys-C or trypsin. The recombinant type A botulinum toxin has the same amino acid sequence as that of type A botulinum toxin prepared from wild-type strain Clostridium botulinum. The sequence of the light chain of recombinant botulinum toxin type A is shown in SEQ ID NO.11, and the sequence of the heavy chain of recombinant botulinum toxin type A is shown in SEQ ID NO.3; Specifically, 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 type A botulinum toxin through recombinant expression, characterized in that, The recombinant botulinum toxin type A as described in any one of claims 1-5 is expressed using a microbial expression system; preferably, the microbial expression system is a prokaryotic expression system; preferably, the prokaryote 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. A method for preparing type A botulinum toxin through recombinant expression according to claim 8, characterized in that, The microbial expression system is incubated with an expression vector, which is formed by integrating a nucleotide fragment of a single-chain protein of botulinum toxin type A with an empty vector. The empty vector includes, but is not limited to, pET-26b, pET-30a, and pET-22a vectors. After fermentation and expression in the microbial expression system and cell disruption, a supernatant containing the target protein is obtained. The supernatant is purified by affinity column chromatography, cation exchange chromatography, enzyme digestion, and cation exchange chromatography to obtain high-quality recombinant botulinum toxin type A.

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

10. The use of the recombinant botulinum toxin type A according to claim 5 in the preparation of drugs for treating nervous system diseases, or in the preparation of drugs for treating muscle spasm diseases, or in the preparation of drugs for relieving pain, or in the preparation of cosmetic products.

Images