Recombinant botulinum toxin and methods of making the same

Abstract

The present application relates to the preparation method of type A recombinant botulinum toxin technical field, specifically relates to the type A botulinum toxin obtained by gene recombination technology, the amino acid sequence is identical with the sequence of type A botulinum toxin prepared by wild strain Clostridium botulinum extraction and purification.The technical scheme provides a single chain protein of type A botulinum toxin, which comprises in sequence: type A botulinum toxin light chain or modified type A botulinum toxin light chain, connecting region, type A botulinum toxin heavy chain.The single chain protein of the scheme is obtained after fermentation, crude purification, enzyme cutting and fine purification High-quality recombinant type A botulinum toxin.The technical scheme can solve the problems of complex process, long cycle, high safety risk and the like of the existing type A botulinum toxin prepared by wild strain Clostridium botulinum, and solve the problem that the existing gene recombination technology cannot obtain the amino acid sequence identical with the type A botulinum toxin prepared by wild strain Clostridium botulinum.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of recombinant botulinum toxin preparation method, and particularly relates to type A botulinum toxin obtained by using gene recombination technology, which has the same amino acid sequence as type A botulinum toxin prepared by extracting and purifying wild Clostridium botulinum. BACKGROUND

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

[0003] In the prior art, the preparation methods mainly include the following two methods:

[0004] One is to obtain by extracting and preparing wild Clostridium botulinum culture, which is the main method for preparing the currently marketed type A botulinum toxin products such as Botox, Xeomin, Dysport and the like. When type A botulinum toxin is prepared by using Clostridium botulinum, the laboratory and preparation process have high requirements due to safety problems, and the preparation process is long and complex. Wild Clostridium botulinum also produces hemagglutinin (HA) and non-hemagglutinin (NTNH) proteins, which may cause adverse reactions such as coagulation and allergy.

[0005] The other is to obtain type A botulinum toxin by gene recombination technology, that is, to construct the type A botulinum toxin protein gene in an expression system by using recombinant protein technology, and to obtain by fermentation and purification. However, it is found from the existing literature that it is difficult to obtain a product with the same amino acid structure as type A botulinum toxin produced by wild Clostridium botulinum by using this technology, and therefore, the safety and effectiveness of the product obtained by the above method need to be re-evaluated and evaluated for a long time.

[0006] Patent CN114989271A (Preparation method of recombinant type A botulinum toxin) uses genetic recombination technology to express light chain protein and heavy chain protein of type A botulinum toxin respectively; the light chain protein is subjected to first denaturation treatment to obtain a first denaturation product; the heavy chain protein is subjected to second denaturation treatment to obtain a second denaturation product; the first denaturation product and the second denaturation product are mixed in a certain proportion, subjected to renaturation and assembly treatment to obtain the BoNT / A. This method for preparing BoNT / A requires additional denaturation and renaturation treatment, which may cause disulfide bond mismatch, and the process steps are relatively complicated and cumbersome, and the yield is not high. Moreover, the N-terminal first amino acid of the light chain and the heavy chain is methionine (M), which is inconsistent with the amino acid structure of BoNT / A obtained by culturing and extracting wild-type Clostridium botulinum.

[0007] Patents CN114957482B (Single-chain polypeptide of modified neurotoxin and use thereof) and CN118006523A (Recombinant genetically engineered bacteria of type A botulinum toxin and preparation and application thereof) also use genetic engineering recombination technology to prepare type A botulinum toxin, but the amino acid structure is inconsistent with that of type A botulinum toxin obtained by culturing and extracting wild-type Clostridium botulinum. More specifically, the natural extraction of type A botulinum toxin is as follows: the sequence of the N-terminal of the heavy chain (HC-N) is ALNDLCIK, the sequence of the N-terminal of the light chain (LC-N) is PFVNKQFN, and the sequence of the C-terminal of the light chain (LC-C) is LLCVRGIITSK. In patent CN118006523A, the above sequence is as follows: the sequence of the N-terminal of the heavy chain (HC-N) is GSK ALNDLCIK, the sequence of the N-terminal of the light chain (LC-N) is GS PFVNKQFN, and the sequence of the C-terminal of the light chain (LC-C) is LLCVRGIITSK TKSLVPR It can be seen that the amino acid sequence of the recombinant type A botulinum toxin of the prior art is actually different from that of the naturally extracted type A botulinum toxin. In addition, the naturally extracted type A botulinum toxin mentioned here refers to the existing commercial products 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 sequence comparison of N-terminal and C-terminal, please see Figure 1 , Figure 2 and Figure 3 It can be seen that the existing recombinant expression technology cannot obtain type A botulinum toxin consistent with the naturally extracted structure.

[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 —(Linking peptide) c —Protein recognition site;

[0018] Where 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 single-chain protein of recombinant botulinum toxin type A is obtained by Lys-C or trypsin enzyme digestion of the recombinant botulinum toxin type A;

[0023] The recombinant botulinum toxin type A is identical in amino acid sequence to Botox and Xeomin prepared by extraction from wild strain 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] wherein 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 present technical solution also provides 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; 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), Rosetta(DE3); more preferably, the strain of Escherichia coli is BL21(DE3).

[0028] Further, the expression system has an expression vector, which has a nucleotide fragment of the single-chain protein of the botulinum toxin type A integrated into the empty vector, including but not limited to pET-26b, pET-30a, pET-22a vector; the expression of the fermentation of the expression system and the cell disruption, to obtain the supernatant containing the target protein; the supernatant is purified by affinity column, cation exchange chromatography, enzyme cutting, cation exchange chromatography, to obtain high-quality recombinant botulinum toxin type A.

[0029] The technical scheme also provides a preparation of other types of botulinum toxin by using the gene recombination technology of the technical scheme.

[0030] The technical scheme also provides an application of the recombinant botulinum toxin type A in the preparation of a drug for treating nervous system diseases, or in the preparation of a drug for treating muscle spastic diseases, or in the preparation of a drug for relieving pain, or in the preparation of a cosmetic product.

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

[0032] The technical scheme also provides an engineering cell for expressing the single-chain protein of the botulinum toxin type A.

[0033] The technical principle of the technical scheme is that:

[0034] In the technical scheme, the gene recombination technology is adopted, the sequence of the single-chain protein is designed, and the cDNA optimization is performed to express in the E. coli. The newly designed sequence (BoNT / A single-chain polypeptide) sequentially includes a first functional region (light chain) containing a metal ion-dependent protease activity domain, a connection region, and a second functional region (heavy chain) containing a binding domain and a translocation domain. The connection region is between the light chain and the heavy chain, and the connection region can be removed by single enzyme cutting, and the heavy chain and the light chain form the BoNT / A protein. Moreover, the structure of the BoNT / A protein obtained by the technical scheme is consistent with the amino acid structure of the naturally extracted BoNT / A protein. Specifically, the protein sequence of the light chain and the heavy chain of the formed BoNT / A protein is completely consistent with the natural sequence, and no extra amino acid residues are present at the N terminal and the C terminal. The disulfide bond between the light chain and the heavy chain is consistent with the natural BoNT / A protein. Although the existing technology has an operation mode of the engineering cell for expressing the BoNT / A single-chain polypeptide, due to the sequence design problem, the BoNT / A protein product usually has a certain enzyme cutting site sequence or a residual short peptide connection, that is, the final product is not completely consistent with the natural BoNT / A protein amino acid structure. The sequence design of the technical scheme and the use of a specific protease for enzyme cutting ensure that the connection region can be completely removed by enzyme cutting, and the light chain and the heavy chain consistent with the natural amino acid structure are formed.

[0035] The patent uses the constructed strain to produce type A botulinum toxin, and high-quality recombinant type A botulinum toxin is obtained after single-chain protease cutting and purification. The obtained single-chain protein is expressed in E. coli, and the obtained bacterial body is subjected to breaking, Ni column chromatography, cation chromatography, enzyme cutting, cation chromatography and anion chromatography in sequence, so that high-purity BoNT / A protein can be obtained. The above-mentioned multiple steps can complete the process preparation in 2-3 days, thereby improving the production efficiency of the BoNT / A protein.

[0036] The beneficial effects of the present scheme are:

[0037] (1) The preparation of type A botulinum toxin is carried out by using gene recombination expression technology, thereby improving safety.

[0038] (2) Type A botulinum toxin with the same structure and amino acid sequence as natural extraction is obtained.

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

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

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

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

[0043] Figure 4 It is a schematic diagram of the plasmid vector map and the connection process of Example 1.

[0044] Figure 5 It is an SDS PAGE electrophoresis image of Example 2.

[0045] Figure 6 It is a complete molecular weight UV labeling diagram of the test sample of Example 3.

[0046] Figure 7Deconvoluted intact molecular weight profile of the test sample for Example 3.

[0047] Figure 8 Reduced UV-labeled profile of the test sample for Example 3.

[0048] Figure 9 Deconvoluted molecular weight profile of the light chain after reduction of the test sample for Example 3.

[0049] Figure 10 Deconvoluted molecular weight profile of the heavy chain after reduction of the test sample for Example 3.

[0050] Figure 11 Primary and secondary mass spectrometry identification profile of the peptide segment (1 : K12 & : NEM (2) [6 37] + H + ) of the test sample for Example 3.

[0051] Figure 12 Primary and secondary mass spectrometry identification profile of the peptide segment (2: K25 & : NEM [5] + H + ) of the test sample for Example 3.

[0052] Figure 13 Primary and secondary mass spectrometry identification profile of the peptide segment (2: K38 & : NEM

[16] + H + ) of the test sample for Example 3.

[0053] Figure 14 Primary and secondary mass spectrometry identification profile of the peptide segment (2: K44 & : NEM [4] + H + ) of the test sample for Example 3.

[0054] Figure 15 BPI profile of the Lys-C enzymatic digest of the test sample for Example 3.

[0055] Figure 16 Component plot mass spectrometry profile of the Lys-C enzymatic cleavage of the light chain C-terminal peptide segment of the test sample for Example 3.

[0056] Figure 17 Primary identification mass spectrometry profile of the Lys-C enzymatic cleavage of the light chain C-terminal peptide segment of the test sample for Example 3.

[0057] Figure 18 Secondary identification mass spectrometry profile of the Lys-C enzymatic cleavage of the light chain C-terminal peptide segment of the test sample for Example 3.

[0058] Figure 19 Component plot mass spectrometry profile of the Lys-C enzymatic cleavage of the heavy chain C-terminal peptide segment of the test sample for Example 3.

[0059] Figure 20Primary identification mass spectrum of C-terminal peptide of heavy chain of Lys-C digested test sample of Example 3.

[0060] Figure 21 Secondary identification mass spectrum of C-terminal peptide of heavy chain of Lys-C digested test sample of Example 3.

[0061] Figure 22 Near-UV CD spectrum of test sample of Example 3.

[0062] Figure 23 Far-UV CD spectrum of test sample of Example 3.

[0063] Figure 24 Reference graph of DAS score of Example 5. DETAILED DESCRIPTION

[0064] The application will be further described in conjunction with the following examples, but the embodiments of the application are not limited thereto. If not specifically indicated, the technical means used in the following examples and experimental examples are the conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial channels.

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

[0066] The term "tag protein" refers to a kind 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 the A-type botulinum toxin.

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

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

[0070] The term "BoNT / A single chain protein", "A type botulinum toxin single chain protein" refers to a single chain polypeptide containing a heavy chain and a light chain, which is expressed by an engineered bacterium. Subsequently, through proteolytic cleavage and other events, the heavy chain and the light chain are separated to form two polypeptide chains, and are combined by disulfide bond to form "BoNT / A protein".

[0071] The term "BoNT / A protein", "A type botulinum toxin protein" refers to A type botulinum toxin extracted from Clostridium botulinum, which is a protein formed by the connection of heavy chain and light chain by disulfide bond after polypeptide enzyme cutting. The sequence of its heavy chain is SEQ ID NO. 3, and the sequence of its light chain is the sequence formed by removing the C-terminal decapeptide and N-terminal methionine M from SEQ ID NO. 1 (SEQ ID NO. 11).

[0072] The term "Lys-C enzyme" refers to a protease that can specifically hydrolyze the peptide bond at the carboxyl end of lysine (Lys, K) and cut the protein chain at the carboxyl end after the lysine residue.

[0073] The term "cation exchange chromatography" refers to a technique that separates molecules with positive charges from molecules with negative charges on the stationary phase by electrostatic interaction. It is mainly used to separate molecules with positive charges under given pH conditions, such as certain proteins, polypeptides or other biological macromolecules.

[0074] The term "anion exchange chromatography" refers to a separation technique based on the interaction between negatively charged molecules and positively charged ligands on the stationary phase. This chromatography method is mainly used to separate proteins, polypeptides and other biological molecules with dissociable negative charges. At a certain pH value, these molecules will carry a negative charge, so they can be electrostatically attracted to the positively charged groups on the stationary phase.

[0075] The term "affinity chromatography" refers to a chromatography technique based on the specific interaction between biological molecules. This method takes advantage of the high affinity and specific binding between two molecules, such as the interaction between antibodies and their antigens, enzymes and their cofactors, receptors and their ligands, etc. By selecting appropriate ligands and fixing them on the solid support of the chromatography column, it is possible to achieve highly specific enrichment and purification of specific target molecules.

[0076] The term "engineered cell" refers to a cell modified by 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 bacterium" refers to a microorganism modified by genetic engineering technology, usually a bacterium, designed to achieve specific functions or produce specific products.

[0078] The term "competent cell" refers to a cell that has been specially treated to be able to take up and integrate foreign DNA under natural conditions.

[0079] The following will be further described in detail through specific embodiments:

[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), and the light chain and the heavy chain are linked by a pair of disulfide bonds (two cysteines at the C-terminus of the light chain and the N-terminus of the heavy chain form), and the heavy chain has a pair of disulfide bonds (two cysteines at the C-terminus of the heavy chain form). The light chain is the active domain, has zinc-dependent metal endopeptidase activity, and 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 membrane of nerve cells and forming an ion channel on the membrane of endosomes, and the translocation domain is responsible for the translocation of the light chain, which transports the light chain into the cell. When designing the expression vector, the heavy chain and the light chain of BoNT / A are usually integrated into the same expression vector, and after the expression of the fusion protein, the heavy chain and the light chain are separated by enzyme digestion, and the two are in the correct conformation of disulfide bond connection. In the present technical solution, a linker peptide is inserted between the fusion protein of the light chain and the heavy chain, forming 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 C-terminal TKSLDKGYNK of the natural sequence is removed). 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 cysteine at position 430 of SEQ ID NO. 1 or 2 and the cysteine at position 6 of SEQ ID NO. 3 form a disulfide bond):

[0083]

[0084] The modified light chain sequence of the type A botulinum toxin 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 according to the degeneracy of codons to correspond to the nucleotide sequences 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 native BoNT / A protein, termed the second functional region containing the binding and translocation domains, the sequence of the A-type botulinum toxin heavy chain is shown in SEQ ID NO. 3 (the cysteine residues C highlighted in grey in the sequence are amino acid residues used to form disulfide bonds: cysteine at position 787 of SEQ ID NO. 3 and cysteine 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 according to the degeneracy of codons to correspond to a nucleotide sequence suitable for the expression requirements of different engineering bacteria, as long as the sequence of the translation product is SEQ ID NO. 3. In subsequent experiments of the technical solution, the specific nucleotide sequence corresponding to SEQ ID NO. 3 used is (SEQ ID NO. 6):

[0094]

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

[0096] (connecting peptide) a (tag protein polypeptide) b (connecting peptide) c X d Y; wherein a: 1-10; b: 1-2; c: 1-10; d: 0-10. That is, in the connecting sequence (also referred to as linker), there are GS short peptides (glycine-serine, i.e., connecting peptide), histidine tags, GS short peptides, and enzyme cutting related sequences (X d Y) in sequence. Preferably, the sequence of the GS short peptide is selected from GGGGS, GGGS, GGS, GGGQ, or GEQP; and the tag protein polypeptide is preferably a histidine tag selected from HHHHHH or HQHQHQ. The enzyme used in this scheme is specifically Lys-C enzyme or trypsin (preferably Lys-C enzyme), and the cleavage is performed at the carboxy terminus of Y of X d 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 recombinant botulinum toxin type A BoNT / A is BoNT / A-LC— linker— BoNT / A-HC, which is sequentially connected at the head and tail. BoNT / A-LC is a light chain of botulinum toxin type A or a modified light chain of botulinum toxin type A; the linker is a connecting region; the connecting region sequentially includes a connecting peptide, a tag protein polypeptide, a connecting peptide, and a protease recognition site (see the foregoing); and BoNT / A-HC is a heavy chain of botulinum toxin type A. The single-chain protein conforming to the structure of “BoNT / A-LC— linker— BoNT / A-HC” can form botulinum toxin BoNT / A with the same amino acid sequence and natural structure after the construction of an expression vector, the construction of an expression bacterium, protein expression, chromatographic purification, enzyme cutting by Lys-C enzyme or trypsin, and chromatographic purification (the preparation process is described in the following text). That is, the sequence of the heavy chain and the light chain of the formed botulinum toxin product is the same as that of commercial products Botox and Xeomin. In addition, in the process of enzyme cutting, the required amount of Lys-C enzyme 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 (mass of Lys-C enzyme / mass of botulinum toxin protein).

[0098] In the following embodiments, the single-chain polypeptide with the sequence of SEQ ID NO. 7 is specifically used to illustrate the experiments, and the sequence of SEQ ID NO. 7 is specifically as follows:

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

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

[0101] GGCGGAGGTGGCTCTGGAGGCGGTGGATCTGGTGGTGGCGGATCACACCACCAC CATCACCACGGTGGAGGCGGGAGCGGAGGCGGCGGTAGTGGTGGAGGAGGCTCTCCC AAG.

[0102] The nucleotide fragment of the BoNT / A single-chain protein is added with enzyme cutting sites at both ends by the conventional means of the prior art, and then integrated into the multiple cloning site of the empty expression vector (select the appropriate enzyme cutting site as needed). The expression vector for expressing the BoNT / A protein precursor formed is transferred into the competent cells to form the engineering cells (engineering bacteria), and the BoNT / A single-chain protein can be obtained through the protein expression of the engineering cells, which is the conventional operation means of the prior art. In the subsequent experimental operation, the Hind III and Nde I enzyme cutting sites are inserted at both ends of the nucleotide fragment of the BoNT / A single-chain protein, and then the nucleotide fragment of the BoNT / A single-chain protein is integrated into the commercial pET-26b empty expression vector through the Hind III and Nde I enzyme cutting sites by the enzyme cutting and ligation method of the prior art, that is, the expression vector for expressing the BoNT / A single-chain protein (pET-26b-BoNT / A) is obtained. The plasmid map and the schematic diagram of the ligation process are shown in detail in Figure 4 The expression vector is transferred into E. coli, and the engineering bacteria for expressing the recombinant BoNT / A protein (BoNT / A protein precursor) are obtained through the conventional transformation and screening. The engineering bacteria specifically selected in the subsequent experimental research are E. coli BL21 (DE3). The strains of E. coli include but are not limited to BL21 (DE3), BL21 (DE3) plysS, JM109 (DE3), Rosetta (DE3); and more preferably, the strain of E. coli is BL21 (DE3). The above-mentioned methods are all the conventional molecular cloning means (recombinant technology) of the prior art, which can be referred to in the tool books such as “Molecular Cloning Experimental Guide”, and will not be described here.

[0103] Example 2: Expression and protein purification of recombinant BoNT / A

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

[0105] The obtained bacteria were subjected to cell disruption, Ni column chromatography, cation exchange chromatography, enzyme digestion, and cation exchange chromatography in sequence. Except for the specific Lys-C enzyme used in the enzyme digestion step, the above processes were conventional protein extraction and purification processes from bacteria in the prior art. The specific processing procedures are described as follows:

[0106] (1) Bacterial collection: The fermentation broth was centrifuged at 7500 r / min for 10 min in a refrigerated centrifuge, and the bacteria were collected. The centrifuged bacteria were stored at -20°C.

[0107] (2) Bacterial dispersion and cell disruption: The bacteria were suspended by stirring with a cell disruption solution (PBS) at a concentration of 100g / L. After ultrasonic cell disruption, the supernatant was collected by centrifugation at 7500 r / min for 10 min at 4°C.

[0108] (3) Ni column purification:

[0109] The reagents used include: equilibration buffer A: 20mM sodium phosphate, pH 7.0; and elution buffer B: 20mM sodium phosphate, 0.3M imidazole, pH 7.0.

[0110] The collected supernatant was loaded into a pre-equilibrated Ni column (Monmix MC60-NTA Ni). After re-equilibration with equilibration buffer A, the fusion protein (BoNT / A single-chain protein) with a histidine tag was separated from other proteins using elution buffer B. The peak containing the BoNT / A single-chain protein was collected to obtain the Ni column purification product. Ni column purification mainly separates the fusion protein (BoNT / A single-chain protein) with a histidine tag from other proteins.

[0111] (4) Cation exchange chromatography:

[0112] The reagents used include: equilibration buffer A: 20mM sodium phosphate, pH 6.5; and elution buffer B: 20mM sodium phosphate, 0.5M sodium chloride, pH 6.5.

[0113] The Ni column purification product obtained by recovering the Ni column was loaded into a 30S column (BestPoly 30S) previously equilibrated with equilibration liquid A, and after re-equilibration, elution was performed using elution liquid B according to a conventional method, and the target peak containing the BoNT / A single-chain protein was collected to obtain the first ion exchange purification product. The target protein and other impurities were further separated by cation exchange chromatography.

[0114] (5) Enzymatic cleavage:

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

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

[0117] By enzyme cleavage, the BoNT / A single-chain protein is cleaved into a heavy chain and a light chain. The sequences of the heavy chain and the light chain are consistent with the sequences of the light chain and the heavy chain in the native BoNT / A protein double chain, and the linking peptide in the middle is removed by enzyme cleavage. The heavy chain and the light chain automatically form a conformationally correct BoNT / A protein. The sequence design of the BoNT / A single-chain protein and the enzyme cleavage method used in this scheme are: the enzyme cleavage positions are "……TSK↓ TKSLDKGYNK ……" and "……GGGGSPK↓ALNDL". By enzyme cleavage, the entire linking region "GGGGSGGGGSGGGGSHHHHHHGGGGSGGGGSGGGGSPK" and the decapeptide "TKSLDKGYNK" at the C-terminus of the first functional region (light chain) are removed. The sequences of the remaining light chain and heavy chain are consistent with the sequences of the light chain and the heavy chain of the native BoNT / A protein. In this scheme, enzyme cleavage occurs at two specific lysine sites of the BoNT / A single-chain protein. Although the BoNT / A single-chain protein also has other lysine sites, the two lysine sites form a specific structure (incorrect structure relative to the native protein) that is easily recognized by Lys-C enzyme and undergoes enzyme cleavage, while enzyme cleavage does not occur at other lysine sites, 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 a native structure BoNT / A protein is obtained.

[0118] (6) Cation exchange chromatography:

[0119] The reagents used include: equilibration 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.

[0120] The enzyme-digested sample was diluted and adjusted to pH 6.5, and then loaded onto a pre-equilibrated 30S column (BestPoly 30S). After re-equilibration with buffer A, the sample was eluted with buffer B, and the peak containing the BoNT / A protein was collected. The cation exchange chromatography further removed non-relevant proteins. The collected product was the BoNT / A final product, i.e., the stock solution. The eluted sample was subjected to SDS-PAGE electrophoresis, and the results are shown in Figure 5 Figure 5 In Figure 1A, from left to right, lane 1 is the non-reduced single-chain protein before enzyme digestion; lane 2 is the reduced single-chain protein before enzyme digestion; lane 3 is the Marker; lane 4 is the stock solution non-reduced (BoNT / A protein); and lane 5 is the stock solution reduced (BoNT / A protein separated into heavy and light chains after reduction). Figure 5 Figure 1B is the SDS-PAGE electrophoresis results of the final stock solution obtained, from left to right, lane 4 is the stock solution non-reduced (BoNT / A protein); lane 2 is the Marker; and lane 3 is the stock solution reduced (BoNT / A protein separated into heavy and light chains after reduction). The Lys-C residue of the stock solution prepared in this example was detected by Elisa, and the residue was less than 0.1 ng / mg (Lys-C enzyme mass / botulinum toxin protein mass).

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

[0122] (1) Complete molecular weight analysis

[0123] Mass spectrometry conditions: The test sample was subjected to mass spectrometry analysis using an Xevo G2-XS QTof mass spectrometer, and the analysis time was 15 min.

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

[0125] The test sample was separated using an ultra-high performance liquid chromatography system. Liquid A was 0.1% formic acid in water, and liquid B was 0.1% formic acid in acetonitrile. The chromatographic column was equilibrated with liquid A. The test sample was loaded onto the automatic sampler, and then separated by the chromatographic column, with a flow rate of 0.3 mL / min, a detection wavelength of 280 nm, and a column temperature of 80°C.

[0126] The complete protein molecular weight was measured to be 148172.9 Da by liquid chromatography-mass spectrometry detection and software analysis. The complete molecular weight UV label chart of the test sample is shown in Figure 6 ; and the deconvoluted complete molecular weight column chart of the test sample is shown in​Figure 7 .

[0127] (2) Reduction molecular weight analysis

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

[0129] After liquid chromatography-mass spectrometry detection and software analysis, the molecular weight of the reduced light chain protein is measured as 50024.4Da, and the molecular weight of the heavy chain is measured as 98151.6Da. The UV label diagram of the molecular weight of the test sample after reduction is shown in Figure 8 ; the deconvolution complete molecular weight column chromatogram of the light chain of the test sample after reduction is shown in Figure 9 ; and the deconvolution complete molecular weight column chromatogram of the heavy chain of the test sample after reduction is shown in Figure 10 .

[0130] The natural BoNT / A protein recorded in Chinese patent CN114957482B: The light chain of the natural BoNT / A has a natural loop region at its C-terminal, which will be cut by an unidentified intracellular peptidase of the clostridium at the latest when the toxin is released from the clostridium during cell lysis under natural conditions. For BoNT / A, the loop sequence on the light chain can be cut off a decapeptide (i.e. the aforementioned TKSLDKGYNK). The cut light chain and heavy chain are connected by a disulfide bond at the N-terminal and C-terminal through a cysteine residue.

[0131] The sequence of the light chain in the natural active BoNT / A finished product (dimer formed by the heavy chain and the light chain) is actually the sequence of SEQ ID NO. 1 minus the C-terminal TKSLDKGYNK and the N-terminal M (the E. coli carries a methionine aminopeptidase, and the initial M is automatically removed after the E. coli expresses the protein, without the need for other operations). The theoretical molecular weight is about 50024Da (i.e. SEQ ID NO. 11, see below). The theoretical molecular weight of the heavy chain of the natural BoNT / A is about 98151Da (i.e. SEQ ID NO. 3).

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

[0133] Botox can be seen at https: / / www.genome.jp / entry / D08957;

[0134] Xeomin can be found at https: / / www.pmda.go.jp / drugs / 2020 / P20200703001 / .

[0135] The heavy chain sequence of the naturally extracted type A botulinum toxin (a light chain and a heavy chain dimer 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:

[0136] PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK.

[0137] The light chain of BoNT / A is obtained by plasmid construction, expression of engineered bacteria, enzyme digestion and purification, and the molecular weight of the light chain is consistent with that of the naturally extracted light chain of BoNT / A double chain. The heavy chain of BoNT / A is obtained, and the molecular weight of the heavy chain is basically consistent with that of the naturally extracted heavy chain of BoNT / A double chain. Through the sequence design and enzyme digestion of the single chain protein in this scheme, all the connecting peptides between the heavy chain and the light chain are removed, and the light chain and the 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, it is replaced into 20mM PB buffer. Part of it is added to Lys-C, and the enzyme digestion reaction is carried out at 37℃ for 18h. 7M Gdn-HCl acidification terminates the reaction, part of the enzyme digestion product is added with 1M TCEP reduction. The enzyme digestion product of the test sample is separated by liquid phase system.

[0140] The test product enzymatic product was desalted and separated by high performance liquid chromatography, and then detected and scanned by a high-resolution mass spectrometer. Analysis time: 120 min, detection mode: positive ion (MSE), parent ion scanning range: 300-2000 m / z.

[0141] After non-reducing enzymatic digestion and liquid chromatography detection analysis, 2 pairs of disulfide bonds consistent with the theoretical disulfide bond pairing mode were identified, namely C429-C443 and C1224-C1269. From the perspective of BoNT / A product (the light chain and the heavy chain have been cleaved and combined by disulfide bond), the 429th amino acid of the light chain is cysteine, the 6th amino acid of the heavy chain is cysteine, and a disulfide bond is formed between them (LC429 and HC6); the 787th amino acid of the heavy chain is cysteine, and the 832nd amino acid is cysteine, and a disulfide bond is formed between them (HC787 and HC832). The formation of disulfide bonds is consistent with the naturally extracted BONT / A.

[0142] The test product was diluted with 20 mM PB solution for freeze-drying concentration, denatured and alkylated by NEM after 20 mM PB buffer was replaced. Part of it was added with Lys-C, and the enzymatic reaction was carried out at 37°C for 18h. The reaction was terminated by 7M Gdn-HCl acidification, and part of the enzymatic product was added with 1M TCEP for reduction. The test product enzymatic product was desalted and separated by high performance liquid chromatography, and then detected and scanned by a high-resolution mass spectrometer. Analysis time: 120 min, detection mode: positive ion (MSE), parent ion scanning range: 300-2000 m / z. The mass spectrometry data was searched and verified by UNIFI (Waters) software for theoretical free thiol peptide data after reduction.

[0143] The alkylated modified peptide segment identification results of the test product are shown in Table 1, and the free thiol ratio of the test product is shown in Table 2. The primary and secondary mass spectrometry identification maps of the test product peptide segment (1: K12 &: NEM (2)

[637] + H + ) are shown in Figure 11 , the primary and secondary mass spectrometry identification maps of the test product peptide segment (2: K25 &: NEM [5] + H + ) are shown in Figure 12 , the primary and secondary mass spectrometry identification maps of the test product peptide segment (2: K38 &: NEM

[16] + H + ) are shown in Figure 13 , the primary and secondary mass spectrometry identification maps of the test product peptide segment (2: K44 &: NEM [4] + H + ) are shown in Figure 14 .

[0144] Table 1: Test product alkylated modified peptide segment identification results (including: Observed retention time (Observed RT), expected mass (Expected mass), observed mass (Observed mass), mass error (Mass Error), etc.)

[0145]

[0146] Table 2: Test product free thiol ratio

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

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

[0148] After analysis, the test product was alkylated by NEM on the free thiol, then digested and reduced, and detected by liquid chromatography. The cysteine was mainly in the free state, with a content of 100% in C133, 100% in C164, 100% in C780, 100% in C956, and 100% in C1049.

[0149] (4) N-terminal sequence analysis

[0150] Electrophoresis: Take enough test product and mix with reduced sample buffer, then boil. Load the test product into two wells, and add pre-stained marker. Concentrate the gel at 100V for 15min, and separate the gel at 180V for 30min.

[0151] Transfer membrane: Prepare PVDF membrane, cut it to the same size as the gel, and evenly wet it in 100% methanol. Place the gel on thick filter paper also soaked in transfer buffer, then cover the PVDF membrane on the gel, cover it with a wet thick filter paper, squeeze out the air to fill it with liquid, and finally clamp them together into the electrotransfer slot. Place the transfer clamp in the transfer slot with the gel side facing the negative terminal, add transfer buffer, open the external circulation to cool to 10°C, and turn on the magnetic stirrer under the electrotransfer slot, adjust the stirrer to low speed. Turn on the power, wet transfer, 250mA for 90min. After the power is turned off, take out the transfer clamp, remove the PVDF membrane and stain it with dye, then rinse it with purified water until the bands are clear. Scan the photo and upload it, and cut out the desired bands to dry the test sample.

[0152] Machine testing:

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

[0154] Data and atlas processing:

[0155] The raw data and the atlas generated by PPSQ are identified by PPSQ Data Processing software and the corresponding atlas is exported.

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

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

[0158] (A-L-N-D-L-X-I-K-V-N-N-W-D-L-F, Cys corresponds to X, 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] (P-F-V-N-K-Q-F-N-Y-K-D-P-V-N-G)

[0161] As can be seen, the N-terminal of the heavy chain and the light chain of the BoNT / A prepared by the scheme is consistent with the sequence of the naturally extracted BoNT / A, without inserting extra amino acid residues or deleting amino acid residues. By using the technical scheme, the sequence of the N-terminal of the heavy chain is completely consistent with SEQ ID NO. 3, and the sequence of the N-terminal of the light chain is completely consistent with SEQ ID NO. 5, without undesired intermediate products. By using the enzyme cutting mode of the scheme, other non-natural heavy chain component amino acid residues will not be introduced at the N-terminal of the heavy chain.

[0162] (5) C-terminal analysis

[0163] Test sample digestion: The test sample was diluted with 20mM PB solution, and an equal volume of 8M UA was added, and denatured at 37℃ for 1h. Lys-C was added, and the enzyme digestion reaction was carried out at 37℃ for 18h, and the reaction was terminated by adding 7M Gdn-HCl acidification, and 1M TCEP reduction was added.

[0164] Liquid chromatography identification: the enzyme digestion sample was separated by UPLC liquid phase system (ACQUITY UPLC I-Class).

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

[0166] Mass spectrometry data processing: Mass spectrometry data was processed using UNIFI control program (1.9.4, Waters), and the theoretical sequence of the test sample was selected as the database for database matching search. The peptide segment information obtained by Lys-C enzyme digestion of the test sample was detected by liquid chromatography-mass spectrometry. The data were searched and matched with the theoretical sequence database and analyzed to identify the C-terminal sequence peptide segments of the light chain and the heavy chain.

[0167] Lys-C enzyme digestion product mass spectrometry detection BPI spectrum is shown in Figure 15 , the component plot mass spectrum of the C-terminal peptide segment of the light chain of the test sample Lys-C enzyme digestion is shown in Figure 16 , the primary identification mass spectrum of the C-terminal peptide segment of the light chain of the test sample Lys-C enzyme digestion is shown in Figure 17 , the secondary identification mass spectrum of the C-terminal peptide segment of the light chain of the test sample Lys-C enzyme digestion is shown in Figure 18 , the component plot mass spectrum of the C-terminal peptide segment of the heavy chain of the test sample Lys-C enzyme digestion is shown in Figure 19 , the primary identification mass spectrum of the C-terminal peptide segment of the heavy chain of the test sample Lys-C enzyme digestion is shown in Figure 20 , the secondary identification mass spectrum of the C-terminal peptide segment of the heavy chain of the test sample Lys-C enzyme digestion is shown in Figure 21 .

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

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

[0170] The C-terminal sequences of the heavy chain and the light chain are consistent with the theoretical sequences, i.e., the BoNT / A protein prepared by the present scheme is consistent with the sequence of the natural BoNT / A protein (molecular weight, C-terminal and N-terminal are 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 by the present scheme is consistent with the natural BoNT / A in terms of amino acid sequence (primary structure), light chain and heavy chain composition, and disulfide bond formation. Therefore, the preparation method of the present technical scheme can obtain a double-stranded form of botulinum toxin BoNT / A that is consistent with the structure of natural endotoxin.

[0172] (6) Circular dichroism analysis

[0173] After the cuvette was soaked in 2M HNO3 overnight and washed with deionized water, it was dried, and then the background and blank buffer were scanned in turn. Then, the appropriate amount of test sample was added to the cuvette for near-ultraviolet (250-340 nm) and far-ultraviolet scanning (190-260 nm). The near-ultraviolet CD spectrum of the test sample is shown in detail in Figure 22 , and the far-ultraviolet CD spectrum of the test sample is shown in detail in Figure 23 .

[0174] The Pro-Data Viewer software was used to process the scanned spectrum by averaging and smoothing, and the smoothing number was set to 3. The ratio of the peak and valley CD values of the standard was calculated, and the effective ratio range was 2.08±0.06. The CDNN software was used to predict the secondary structure of the spectrum.

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

[0176] Table 3: Secondary structure prediction statistical table of each test sample

[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 (crude solution virulence)

[0179] (1) Experimental design and grouping:

[0180] After the 26-30 day old male Kunming mice (SPF level) were adaptively fed, 60 mice with similar body weights were selected and randomly divided into 6 groups, 10 mice in each group. The samples were used to prepare the test samples, and the samples were diluted to the same concentration. After administration, the toxicity reactions of the mice and the death of the animals in each group were closely observed for 4 consecutive days. The sample was the sample prepared in Example 2, and the eluate collected after cation exchange chromatography in "(6) Cation exchange chromatography" was called the crude solution.

[0181] (2) Experimental process:

[0182] Before the experiment, the experimental mice were weighed, and the body weight was evenly distributed to each group to ensure that the average body weight of the animals in each group had no statistically significant difference. The experiment was divided into 6 groups, 10 mice in each group, and the mice were male. Intraperitoneal injection was used for administration; one person drew the test sample solution, and another person checked and completed the animal administration operation, and recorded the administration time after injection. The grouping, administration dose information of the test sample A type botulinum toxin is shown in Table 4.

[0183] 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.

[0184] Table 4: Results of toxicity test of BONT / A protein obtained in Example 2

[0185]

[0186]

[0187] Example 5: DAS scoring

[0188] Forty healthy, sexually mature female SD rats (SPF grade), weighing approximately 180-200g, were randomly divided into four groups (n=10 per group) after acclimatization: a solvent control group, a low-dose group, a medium-dose group, and a 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 scoring criteria). Figure 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 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 drug administration (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.

[0190] Table 5: Experimental grouping and drug administration arrangements

[0191]

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

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

[0194]

[0195]

[0196] Comparative Example 1

[0197] In the prior art, endotoxins with a natural structure are mainly obtained in two ways: extracted from Clostridium botulinum; the heavy chain and the light chain are expressed in different engineering bacteria respectively, and then assembled to obtain the botulinum toxin.

[0198] In the prior art, attempts have also been made to integrate the heavy chain and the light chain of botulinum toxin to form a fusion protein, express in the same engineering bacteria, and then obtain botulinum toxin through enzyme cutting and other operation steps. For example, Chinese patent CN114957482B (a single-chain polypeptide of a modified neurotoxin and uses thereof) links the tag protein, the light chain and the heavy chain in sequence to form a fusion protein, and sets enzyme cutting 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 the patent technology is: tag protein, first protease cutting site LEVLFQGPL, connecting short peptide GS, light chain of first functional amino acid structure region BoNT / A (C-terminal: ……CVRGIITS, when designing the sequence, the amino acid residues of the C-terminal loop region are removed in advance), second protease cutting site LEVLFQGP and heavy chain of second functional amino acid structure region BONT / A (N-terminal: ALNDLCIK…). After the recognition sequence LEVLFQGP of Tobacco Etch Virus (TEV) protease is cut (LEVLFQ↓GP), the light chain and the heavy chain are recombined to form a dimer (double-chain botulinum toxin). However, the above-mentioned method cannot obtain botulinum toxin with a natural structure. For example: the light chain sequence is not a natural sequence, and it still has the residues of the enzyme cutting site at its C-terminal and at its N-terminal, as well as the GS short peptide. For another example, the heavy chain is also not a natural sequence, and it still has the amino acid residues of the enzyme cutting site at its N-terminal. Since the sequence of the botulinum toxin obtained by the patent technology has the amino acid residues of the non-natural botulinum toxin, the botulinum toxin obtained by the method is also not a botulinum toxin with a natural structure, which is different from the technical scheme. The sequence alignment can be seen in Figures 1-3 .

[0199] Chinese patent CN118006523A (Recombinant genetically engineered bacteria of botulinum toxin type A and preparation and application thereof) also attempts to produce recombinant botulinum toxin type A. The specific scheme is as follows: using pET-28a vector to integrate recombinant single-chain polypeptide of botulinum toxin, then performing protein expression, cutting and purification, and finally forming botulinum toxin type A. Among them, the recombinant single-chain polypeptide includes gene fragment 1 expressing GST tag, gene fragment 2 expressing thrombin recognition site, and gene fragment 3 expressing botulinum toxin type A BoNT / A; the light chain and heavy chain connection region of BoNT / A includes another thrombin recognition site and the amino acid sequence of GST. The thrombin recognition site is LVPRGS, and the cutting position is LVPR↓GS. Similarly, the N-terminal of the heavy chain of BONT / A formed by cutting according to the above method, or the N-terminal and C-terminal of the light chain, will leave some non-natural BONT / A amino acid residues, resulting in that the overall product (especially the primary structure of the polypeptide) is not natural structure. Sequence alignment can be seen in Figures 1-3 .

[0200] The special design of the BONT / A precursor (single-chain polypeptide) in this scheme can ensure that the heavy chain and light chain of BONT / A with the same structure as naturally extracted are obtained after enzyme cutting, and the overall product is consistent with 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 scheme can not only improve the efficiency of the preparation process, but also ensure that the product structure is completely consistent with the naturally extracted structure. Compared with the prior art of preparing BoNT / A by recombinant protein single-chain polypeptide, it has more advantages.

[0201] Comparative Example 2

[0202] The prior art usually expresses the light chain and the heavy chain separately, and then combines the light chain and the heavy chain to form BoNT / A. For example, in Chinese patent CN115894641B (Construction of botulinum toxin type A mutant and genetically engineered bacteria thereof), the light chain and the heavy chain are expressed separately, and the cysteines of the light chain and the heavy chain are mutated. However, the light chain and the heavy chain are expressed separately, which will increase the process flow and complexity to some extent. Moreover, the product obtained by mutating the light chain and the heavy chain in this scheme has a large structural difference from the naturally extracted product, so its safety and effectiveness need to be further studied. Compared with the product of this scheme which has the same structure as the naturally extracted BoNT / A, it is more difficult and takes a longer period to realize the clinical application of the product.

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

[0204] The present comparative example explores a large number of enzyme cutting sites. First, the enzyme cutting site is set at the N-terminus of the single-chain protein. The amino acid sequence of the single-chain protein is shown as follows (SEQ ID NO. 9):

[0205]

[0206]

[0207] In the above sequence, a histidine tag and a connecting sequence (indicated by double lines) are added at the N-terminus of the light chain. 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 the present application. An enzyme cutting site "AEAEAPK↓PFVNKQFNY" is set at the N-terminus of the light chain, and it is planned to be cut at this site. After cutting, the N-terminus of the light chain is consistent with the natural sequence. Referring to the methods of Example 1 and Example 2, BoNT / A is prepared, except that the sequence of the single-chain protein is replaced by SEQ ID NO. 9, and the enzyme cutting method is performed according to the preferred method of Example 2. The N-terminus / C-terminus of the light chain and the heavy chain of the obtained botulinum toxin BoNT / A is sequenced, the N-terminus of the light chain is SDKII (not cut at the designed enzyme cutting site), the N-terminus of the heavy chain is ALNDL (consistent with the natural sequence), and the C-terminus is consistent with the natural sequence. The sequencing results show that the N-terminus of the light chain is difficult to form the enzyme cutting method expected by the designed sequence, and the structure of the obtained botulinum toxin is inconsistent with the natural botulinum toxin. Therefore, it is not appropriate to add a tag at the N-terminus of the single-chain protein.

[0208] Then, the enzyme cutting site is set at the C-terminus of the single-chain protein. The amino acid sequence of the single-chain protein is shown as follows (SEQ ID NO. 10):

[0209]

[0210]

[0211] In the above sequence, a histidine tag and a connecting sequence (indicated by double line) are added to the C-terminal of the heavy chain, and the light chain and the heavy chain are directly connected by a decapeptide (in the natural form), without setting a connecting peptide containing a histidine tag between the two as in the present application. The BoNT / A is prepared according to the method of Example 1 and Example 2, except that the single-chain protein sequence is replaced by SEQ ID NO. 10, and the enzyme cutting is performed according to the preferred method of Example 2. The molecular weight of the light chain and the heavy chain of the obtained botulinum toxin BoNT / A is detected (reduced molecular weight), and the molecular weight of the light chain is 50025.8, and the molecular weight of the heavy chain is in multiple cases: 98280.60, 95564.5, 95039.0. The results show that the light chain is consistent with the theory, and the heavy chain is not completely cut, and the error cutting occurs. The molecular weight detection result shows that according to the design of the single-chain protein sequence described above, the C-terminal of the heavy chain is difficult to form the expected enzyme cutting mode of the designed sequence, and the structure of the obtained botulinum toxin is inconsistent with that of the natural botulinum toxin. Therefore, it is not appropriate to add a tag to the C-terminal of the single-chain protein.

[0212] The present technical solution designs a histidine tag between the light chain and the heavy chain, and the addition of the histidine tag facilitates protein purification, and through the enzyme cutting treatment of Lys-C enzyme, a botulinum toxin with a natural structure can be formed, which achieves an unexpected technical effect compared with setting a tag protein at the C-terminal and the N-terminal of the single-chain protein.

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

[0214] The single-chain protein sequence of SEQ ID NO. 10 of Comparative Example 3 cannot correctly cut the C-terminal of the heavy chain by using the enzyme cutting mode of Example 2, but can correctly cut the C-terminal of the light chain and the N-terminal of the heavy chain. The connection between the heavy chain and the light chain of the single-chain protein of 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 by the aforementioned decapeptide).

[0215] More specifically, the cleavage method for the single-chain protein is: pH 8.0, the ratio of 1 g of BoNT / A single-chain protein to 1 mg of Lys-C enzyme (the ratio of enzyme to single-chain protein is 1:1000), 4 h of cleavage time, and 25°C of cleavage temperature. Under this condition, the C-terminal of the light chain and the N-terminal of the heavy chain can form correct cleavage (the obtained product is consistent with the natural sequence). However, the amount of Lys-C enzyme in this cleavage method is still relatively large, which is not conducive to the conservation of raw materials and the reduction of impurities in the final product. The amount of enzyme was tried to be reduced, and the cleavage result was observed again. The cleavage method for the single-chain protein of SEQ ID NO. 10 was modified to: pH 8.0, 1 g of BoNT / A single-chain protein was added to 0.05 mg of Lys-C enzyme (the ratio of enzyme to single-chain protein was 1:20000), and cleavage was performed at 20°C for 16 h. The rest of the operation method was the same as in Example 2, only the cleavage condition was changed, and the cleavage product was prepared by referring to the method of Example 2. The protein in the stock solution was detected, and it was found that a large amount of protein in the stock solution was not cut open, which was specifically reflected in that: after the protein in the stock solution was reduced and treated, electrophoresis was performed, and 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) appeared, indicating that the light chain and the heavy chain were not fully cut open under this cleavage condition. In addition, the reduced molecular weight was performed, and in addition to the correct light chain molecular weight (about 50024 Da) and the heavy chain molecular weight (about 98151 Da), the reduced molecular weight of the light chain and the heavy chain showed multiple forms, either slightly larger than the correct molecular weight or slightly smaller than the correct molecular weight. It is indicated that the light chain and the heavy chain adopt the natural connection method, and to achieve the correct cleavage of the C-terminal of the light chain and the N-terminal of the heavy chain to form a sequence consistent with the natural structure, there are certain requirements for the cleavage method (especially the amount of Lys-C enzyme).

[0216] The single-chain protein sequence (SEQ ID NO. 7) of the present scheme is subjected to enzyme digestion, and the enzyme digestion is as follows: pH 8.0, 1 g of BoNT / A single-chain protein is added to 0.05 mg of Lys-C enzyme at a ratio of 1:20000 for enzyme digestion (the ratio of enzyme to single-chain protein is 1:20000), and the enzyme digestion is performed at 20°C for 16 h. The rest of the operation is the same as in Example 2, only the enzyme digestion conditions are changed, and the enzyme digestion product is prepared into a stock solution according to the method of Reference Example 2. The reduced molecular weight determination is performed, and the results are consistent with the theory, the light chain is about 50024.00, and the heavy chain is about 98151.75, and no other molecular weight forms are present. No complete cutting between the light chain and the heavy chain occurs, and no cutting site error occurs at the C-terminal of the light chain and the N-terminal of the heavy chain. The sequences of the light chain and the heavy chain of the botulinum toxin product obtained are consistent with the natural sequences, and the cutting effect of the increased Lys-C enzyme dosage in Example 2 is consistent, and the botulinum toxin product with the same structure as the natural structure can be formed. The Lys-C residue in the stock solution prepared by the present preparation method is detected by Elisa, and the residue is less than 0.1 ng / mg (Lys-C enzyme mass / botulinum toxin protein mass).

[0217] According to the above experimental results, the present scheme sets a connection 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 connection peptide ensures that the light chain and the heavy chain are correctly cut, and the C-terminal of the light chain and the N-terminal of the heavy chain completely meet the natural structure. And due to the presence of the connection peptide, the amount of enzyme required for enzyme digestion is greatly reduced. Even in the case of a ratio of 1:20000 of enzyme to single-chain protein, the C-terminal of the light chain and the N-terminal of the heavy chain can form a correct cutting form, and there will be no insufficient enzyme cutting or enzyme cutting site error. Therefore, the upstream design of the botulinum toxin product of the present technical scheme is different from the conventional method of the prior art. Through the design of the connection 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 the amount of enzyme used for enzyme cutting of the single-chain polypeptide can be greatly reduced, thereby reducing the cost of reagents, reducing the residue of impurities in the product, and reducing the difficulty of subsequent purification process.

[0218] The above is only an embodiment of the present application, and the common knowledge of the specific structure and characteristics in the scheme is not described in detail. It should be pointed out that for those skilled in the art, without departing from the structure of the present application, a number of modifications and improvements can be made, which should be regarded as the protection scope of the present application, which will not affect the effect and practicality of the patent. The protection scope claimed in the present application should be subject to the content of its claims, and the specific implementation mode and the like recorded in the specification can be used to explain the content of the claims.

Claims

1. A single-chain protein of recombinant botulinum toxin type A, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

7.

2. A method for preparing type A botulinum toxin through recombinant expression, characterized in that, The single-chain protein of the recombinant botulinum toxin type A as described in claim 1 was expressed using a microbial expression system.

3. The method for preparing type A botulinum toxin by recombinant expression according to claim 2, characterized in that, The microbial expression system is a prokaryotic expression system.

4. The method for preparing type A botulinum toxin by recombinant expression according to claim 3, characterized in that, The prokaryote is Escherichia coli.

5. The method for preparing type A botulinum toxin by recombinant expression according to claim 4, characterized in that, The strains of Escherichia coli were selected from BL21 (DE3), JM109 (DE3), and Rosetta (DE3).

6. The method for preparing type A botulinum toxin by recombinant expression according to claim 4, characterized in that, The strain of Escherichia coli was selected from BL21(DE3)plysS.

7. The method for preparing type A botulinum toxin by recombinant expression according to claim 4, 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 into an empty vector. The empty vector is selected from 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 recombinant botulinum toxin type A.

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