Preparation method of recombinant botulinum toxin A

Abstract

The invention relates to the technical field of biology, and particularly discloses a preparation method and application of recombinant A-type botulinum toxin. According to the method disclosed by the invention, after the recombinantly expressed protoxin is combined with cation exchange filler, enzyme digestion and activation are realized by utilizing single protease, and then purification is carried out. The preparation method disclosed by the invention has the advantages of good convenience and small batch-to-batch difference, and the prepared recombinant A-type botulinum toxin also has obvious advantages in protein effectiveness and biological activity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing recombinant type A botulinum toxin and its application. Background Technology

[0002] Botulinum toxin type A (BoNT / A) is a neurotoxin produced by Clostridium botulinum and is widely used in medical aesthetics (such as wrinkle removal and facial slimming) and medical treatments (such as muscle spasms and chronic migraines). Traditional natural botulinum toxin type A is mainly obtained through Clostridium botulinum fermentation, extraction, and purification, which presents problems such as high biosafety risks, poor batch-to-batch consistency, high levels of impurity proteins, large amounts of non-BONT / A components, and high immunogenicity. In recent years, with the development of genetic engineering and recombinant protein technology, recombinant botulinum toxin type A has gradually become a research hotspot. Compared to natural toxins, recombinant technology can express the active toxin protein in non-pathogenic hosts (such as E. coli and insect cells), avoiding the use of highly pathogenic strains, significantly reducing biosafety risks, and improving product purity and consistency.

[0003] Currently, there are three main routes for the preparation of recombinant botulinum toxin:

[0004] One method involves expressing the light chain LC and heavy chain HC separately in *E. coli* as inclusion bodies. After preliminary purification of the inclusion bodies, recombinant BONT / A is obtained through denaturation, renaturation, and assembly, as described in existing patent CN114989271B. The recombinant BONT / A obtained by this method is completely identical to the natural sequence. However, the inclusion body protein requires a complex denaturation and renaturation process to restore its biological activity, and there is a certain risk of misfolding, which may affect safety.

[0005] Secondly, a specific restriction enzyme site is introduced between the light chain LC and the heavy chain HC to solublely express the light chain LC+heavy chain HC single chain in E. coli. After preliminary purification, LC-HC dimer is obtained by enzyme digestion. The light chain LC+heavy chain HC single chain expressed by this method has low toxicity, and soluble expression can maintain the native structure and biological activity of the recombinant protein, which is beneficial for commercial production. However, due to the introduction of exogenous specific restriction enzyme sites, the recombinant BONT / A protein prepared by this method contains exogenous amino acid residues.

[0006] Thirdly, a soluble expression tag protein + light chain LC + heavy chain HC single chain is used. No specific cleavage sites are introduced between the light chain LC and the heavy chain HC. After initial purification of the single-chain protein, enterokinase is used to cleave the tag protein, simultaneously cleaving the light chain LC and the heavy chain to form a dimer, thus activating the toxin, as described in the existing patent CN120350044A. This method does not introduce exogenous cleavage sites; however, due to the low rate of enterokinase action, the cleavage speed is slow, and the cleavage environment parameters are unfavorable to the stability of the activated toxin, leading to incomplete and inconsistent cleavage. The main problem is the inconsistent C-terminal length of the light chain, resulting in poor product consistency. Because the difference between the toxin protein before and after activation is small, it poses a challenge to purify and remove unactivated and over-cleaved proteins after activation. Impurity proteins may remain in the final product, posing a certain risk to safety and efficacy.

[0007] For the soluble expression of single-chain botulinum toxin, the enzymatic activation process is particularly important. Regardless of the activation method used, the termination of enzyme digestion after activation is a key factor. Generally, the estimated end time of enzyme digestion activation needs to be confirmed by SDS-PAGE electrophoresis. Electrophoresis followed by staining and destaining takes several hours. After confirming complete activation, subsequent purification to remove the activating enzyme typically takes 1-2 hours. Therefore, from the start of product activation, it takes 3-4 hours to remove the activating enzyme and truly terminate the digestion. During this period, even with cryoprotection or dilution, the coexistence of the activating enzyme and the toxin protein significantly impacts the stability of the activated toxin protein. Protease inhibitors can also be used to terminate enzyme digestion activation, but their addition increases the difficulty of subsequent purification and removal, and the introduction of one or more process impurities poses a risk to the safety and efficacy of the product.

[0008] More importantly, the method of activating recombinant botulinum toxin single-chain toxin using trypsin digestion has some drawbacks. The natural linker region between the light and heavy chains contains four lysine residues, all of which have a chance of being cleaved, and the cleavage can be inconsistent, resulting in inconsistencies in the amino acid sequence of the final toxin protein. In addition, trypsin has a certain probability of cleaving hidden cleavage sites within the toxin itself, causing degradation or decreased activity of the toxin protein. Therefore, many researchers avoid the natural single-chain sequence and address this issue by introducing specific cleavage sites between the light and heavy chains. However, this method easily leads to the residue of non-natural exogenous amino acids, posing potential risks to drug safety and efficacy.

[0009] Recombinant botulinum toxin type A has extremely high biological activity, with a theoretical specific activity of 1×10⁻⁶. 8For highly active proteins with concentrations above U / mg, improper or delayed enzymatic activation processes can cause significant and irreversible damage to the toxic protein. Therefore, developing a convenient, effective, and easy-to-operate preparation process is crucial. In light of this, this invention is proposed. Summary of the Invention

[0010] To address the problems in the prior art, this invention seeks a novel method for preparing recombinant type A botulinum toxin. This invention involves the soluble expression of a light chain + heavy chain single-chain combination of type A botulinum toxin. After preliminary purification, the toxin is bound to a cation exchange packing material. Following activation with only one protease, the protease is rapidly removed, and the toxin protein is further purified. Furthermore, the activation process binds the protein to the packing material particles, ensuring consistent cleavage endpoints at 438 aa at the C-terminus of the light chain, reducing the risk of erroneous or excessive cleavage and maximizing the preservation of the activated toxin protein's activity.

[0011] Therefore, the present invention includes at least the following objectives:

[0012] The primary objective of this invention is to find a nucleic acid construct suitable for the preparation of recombinant botulinum toxin type A and a corresponding protein expression method;

[0013] The second objective of this invention is to find a novel method for preparing recombinant type A botulinum toxin and its application.

[0014] To achieve the above objectives, the present invention provides the following detailed technical solution:

[0015] The present invention first provides a BONT / A nucleic acid construct, which includes a tag sequence and a BONT / A nucleic acid sequence;

[0016] Furthermore, the tag sequence encodes the MBP tag and the His tag;

[0017] Furthermore, the tag sequence sequentially encodes the MBP tag, the artificial connector sequence, and the His tag;

[0018] In some embodiments, the artificial connector sequence is NNNNNNNNNNNGGGGS.

[0019] In some specific embodiments, the tag sequence is a nucleic acid sequence encoding the protein sequence shown in SEQ ID NO.1. In some more specific embodiments, the nucleic acid sequence of the tag sequence is specifically shown in SEQ ID NO.3.

[0020] Furthermore, the BONT / A nucleic acid sequence encodes a natural BONT / A protein sequence (which contains light and heavy chain sequences), and no restriction enzyme sites are introduced in the sequence (especially the sequence between the light and heavy chains);

[0021] In some specific embodiments, the natural BONT / A protein sequence is shown in SEQ ID NO.2;

[0022] In some more specific embodiments, the BONT / A nucleic acid sequence is shown as in SEQ ID NO.4.

[0023] Furthermore, the BONT / A nucleic acid construct also includes a flexible adapter sequence that connects the tag sequence and the BONT / A nucleic acid sequence;

[0024] In some embodiments, the flexible sequence encodes an amino acid linker, wherein the amino acid linker is any combination of G and S, and the C-terminus of the sequence is R or K;

[0025] In some specific embodiments, the amino acid linker includes, but is not limited to, the following sequences: GGGSR, GGGSK, GSSGSSR, GSSGSSK, GGSGGGSR, or GGSGGGSK.

[0026] The present invention also provides the use of any of the aforementioned BONT / A nucleic acid constructs in the expression or preparation of recombinant botulinum toxin type A.

[0027] This invention also provides a method for preparing recombinant type A botulinum toxin, the method comprising the following steps:

[0028] 1) Recombinant protein expression: The aforementioned BONT / A nucleic acid construct was recombinantly expressed under conditions suitable for protein expression;

[0029] 2) First purification of recombinant protein: The expression product is purified to obtain the first purified product;

[0030] 3) Recombinant protein cleavage activation to obtain activating toxin: The first purified product is combined with the purified packing material and activated with trypsin. After activation, the product is eluted according to a method suitable for the purified packing material to terminate the activation and obtain the activated protein.

[0031] Furthermore, in step 1), the conditions suitable for protein expression include, but are not limited to, the following: inserting the BONT / A nucleic acid construct into a plasmid to construct a recombinant expression vector, constructing the recombinant expression vector into recombinant bacteria, and obtaining a fermentation broth by fermentation culture and protein expression of the recombinant bacteria;

[0032] In some embodiments, the recombinant bacteria may include recombinant Escherichia coli, etc.

[0033] In some specific embodiments, the Escherichia coli may be selected from E. coli BL21(DE3), E. coli C41(DE3), or BL21(DE3)pLysS; preferably selected from E. coli BL21(DE3).

[0034] Furthermore, in step 2), the purification is preferably chromatographic purification, and more preferably cation exchange chromatography purification;

[0035] In some specific methods, the cation exchange chromatography purification can be as follows: select SP Sepharose High Performance (Cytiva) cation exchange chromatography packing material, add protein extraction solution, elute with 50 mM Tris-HCl, pH 7.8 elution buffer A, and then elute the target protein with 20% of 50 mM Tris-HCl, 1 M NaCl, pH 7.8 elution buffer B to obtain the toxin source purified solution.

[0036] Furthermore, in step 3), the purification packing material is a cation exchange chromatography packing material;

[0037] The term "cation exchange chromatography packing material" refers to a material that uses a solid-phase support as a matrix and bonds negatively charged functional groups (such as carboxyl-COOH, sulfonic acid-SO3H, etc.) to specifically bind positively charged target molecules in the target sample through electrostatic interactions. Therefore, the type of cation exchange chromatography packing material described in this invention is not limited, as long as it can achieve the binding of the target cationic protein based on its fundamental binding characteristics, it should also satisfy the steric hindrance effect of this invention, thereby facilitating precise enzymatic cleavage and activation. In some specific embodiments, the cation exchange chromatography packing material is selected from SP Sepharose HighPerformance (Cytiva), Diamond CD-S (Borglon), or SP Sepharose FF (Cytiva), etc.

[0038] Furthermore, the form of use of the cation exchange chromatography described in this invention is not limited; it can be selected from the form of a chromatography column for protein binding or from the form of a suspension for protein binding. Based on the clear core concept of this invention, it can be expected that different forms of use can achieve the corresponding steric hindrance effect, thereby achieving the effect of this invention.

[0039] In some specific embodiments, the cation exchange chromatography packing material of the present invention is SP Sepharose HighPerformance (Cytiva), and the aforementioned step 3) may specifically include the following aspects:

[0040] Select cation exchange chromatography packing material: SP Sepharose High Performance (Cytiva), and prepare the appropriate elution buffer; dilute the first purified product (toxin protein) with the elution buffer and load it onto the packing material to bind the toxin protein to the cation exchange packing material, and then rinse appropriately with the elution buffer; prepare trypsin working solution and perform enzyme digestion and activation by addition; after enzyme digestion and activation, terminate the activation by appropriate elution to obtain the activated protein.

[0041] Some more specific steps may be as follows:

[0042] Select cation exchange chromatography packing material: SP Sepharose High Performance (Cytiva). Prepare elution buffer A: 20 mM Tris-HCl pH 8.2; elution buffer B: 20 ​​mM Tris-HCl + 1 M NaCl pH 8.2. Dilute the first purified product (toxin protein) with elution buffer A to a conductivity of 2-4, and load the sample at a flow rate of 3-6 ml / min to bind the toxin protein to the cation exchange packing material. Elute with elution buffer A for 3-4 column volumes. Based on trypsin activity, prepare a 1 U / ml trypsin working solution by diluting with 20 mM Tris-HCl pH 8.2 buffer containing 2 mM CaCl2. Activate the enzyme by feeding at a flow rate of 1 ml / min for 1 hour. After enzyme digestion, elute with elution buffer A for 3 column volumes, then elute with 15% elution buffer B to obtain the activated protein. Simultaneously, the sample has completed the removal of trypsin and tag protein, so terminate the activation and obtain the activated protein.

[0043] In addition, to obtain high-purity activated toxin protein, the preparation method of the present invention may further include a second purification step and / or a third purification step.

[0044] Furthermore, the second purification can be Ni column affinity purification, thereby removing trace amounts of undigested His-tagged intact protein;

[0045] Furthermore, the third purification can be a conventional molecular sieve purification method in the art, thereby further improving protein purity.

[0046] It is understandable that the second and third purifications are based on the core ideas of this invention, and are aimed at further improving the purity of the active protein.

[0047] Finally, the present invention also provides a recombinant type A botulinum toxin, which is obtained by any of the preparation methods described above.

[0048] The advantages of this invention are as follows: In terms of effectiveness, the light chain + heavy chain single strands of this invention are natural sequences, without any non-natural enzyme cleavage sites introduced in between. Activation is achieved through enzymatic digestion using purified packing material. The physical steric hindrance formed by the binding of the purified packing material and the toxin protein solves the problem of inconsistent C-terminal cleavage of the light chain. Furthermore, the activating enzyme can be quickly removed after activation, immediately terminating the enzymatic digestion. The amino acid sequence of the activated toxin protein is consistent with that of commercially available naturally extracted products. In terms of convenience, the enzyme digestion of the solubilization tag and the activation of the toxin protein in this invention use the same protease. Enzymatic digestion at two sites is performed in the same step, significantly reducing the time the toxin is under enzymatic digestion conditions and effectively improving the toxin activity during production. In terms of protein structure, the soluble expression of this invention ensures the natural structure and biological activity of the recombinant BONT / A protein. In terms of accuracy, this invention ensures complete activation of the toxin protein, resulting in high product consistency. The N / C-terminal amino acid sequences of the light chain LC and heavy chain HC of the toxin protein are consistent with those of natural Clostridium botulinum extracts, with small batch-to-batch variations, which is beneficial for commercial production. In terms of biological activity, the recombinant BONT / A prepared by this invention exhibits extremely high biological activity, maximally protecting the activity of the activated toxin protein, with a specific activity > 5 × 10⁻⁶. 8 Compared with traditional enzyme digestion and process design, the specific activity is increased by 20 times (U / mg). Attached Figure Description

[0049] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0050] Figure 1 Structural prediction diagram of toxinogen protein.

[0051] Figure 2 The electrophoresis results of the first purification solution are shown in the figure. Among them, band 1 is the clear solution, band 2 is the breakthrough solution, band 3 is the eluent, band 4 is the purified toxin source solution after the first step, and band 5 is 100% solution B.

[0052] Figure 3 Figure 1 shows the electrophoresis results of the activated protein; where a is the electrophoresis result of the activated protein of the present invention, and b is the electrophoresis result of the activated protein of the conventional method.

[0053] Figure 4 Figure 2 shows the electrophoresis results of the second purified solution.

[0054] Figure 5 Figure 1 shows the electrophoresis results of the third purification solution.

[0055] Figure 6 The mass spectrometry detection results of the C-terminal sequence of the protein light chain in the original solution of this invention; in the figure, the top, middle and bottom figures are the extracted ion chromatogram, the first-order mass spectrum and the second-order mass spectrum, respectively.

[0056] Figure 7 The mass spectrometry results of the C-terminal sequence of the protein heavy chain in the original solution of this invention; in the figure, the top, middle and bottom figures are the extracted ion chromatogram, the first-order mass spectrum and the second-order mass spectrum, respectively.

[0057] Figure 8 The mass spectrometry detection results of the N-terminal sequence of the protein light chain in the original solution of this invention; in the figure, the top, middle and bottom figures are the extracted ion chromatogram, the first-order mass spectrum and the second-order mass spectrum, respectively.

[0058] Figure 9 The mass spectrometry detection results of the N-terminal sequence of the protein heavy chain in the original solution of this invention; in the figure, the top, middle and bottom figures are the extracted ion chromatogram, the first-order mass spectrum and the second-order mass spectrum, respectively.

[0059] Figure 10 Mass spectrometry results of the C-terminal sequence of the light chain of recombinant purified protein obtained by traditional enzyme digestion method; in the figure, the top, middle and bottom figures are the extracted ion chromatogram, the first-order mass spectrum and the second-order mass spectrum, respectively.

[0060] Figure 11 Mass spectrometry results of the C-terminal sequence of the light chain of recombinant purified protein obtained by traditional enzyme digestion method; in the figure, the top, middle and bottom figures are the extracted ion chromatogram, the first-order mass spectrum and the second-order mass spectrum, respectively. Figure 12 Mass spectrometry results of the N-terminal sequence of the light chain of recombinant purified protein obtained by traditional enzyme digestion methods; in the figure, the top, middle and bottom images are the extracted ion chromatogram, the first-order mass spectrum and the second-order mass spectrum, respectively. Figure 13 Mass spectrometry results of the N-terminal sequence of the light chain of recombinant purified protein obtained by traditional enzyme digestion methods; in the figure, the top, middle and bottom images are the extracted ion chromatogram, the first-order mass spectrum and the second-order mass spectrum, respectively. Figure 14 Figure 1 shows the results of capillary isoelectric focusing electrophoresis detection of the original protein of this invention. Figure 15 Figure 1 shows the SEC-MALS purity test results of the original protein of this invention. Detailed Implementation

[0061] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0062] Definitions of some terms

[0063] Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this invention are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0064] As used in this invention, the indefinite or definite articles used when referring to singular nouns, such as “a” or “a kind”, “the”, include the plural form of the noun.

[0065] As used in this invention, the terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term “consisting of” is considered a preferred embodiment of the term “comprising.” If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of those embodiments.

[0066] The term "approximately" in this invention refers to an accuracy range that, as would be understood by those skilled in the art, still guarantees the technical effects of the features in question. This term typically indicates a deviation from the indicated value of ±10%, preferably ±5%.

[0067] Furthermore, the terms first, second, third, (a), (b), (c), and similar terms used in the specification and claims are for distinguishing similar elements and are not necessary for the order of description or chronological sequence. It should be understood that such terms are interchangeable in appropriate contexts, and the embodiments described in this invention can be implemented in a different order than that described or illustrated in this invention.

[0068] The embodiments of the present invention will be described in detail below with reference to examples. The present invention includes, but is not limited to, the following embodiments. Any modifications made to the present invention based on existing technology that do not depart from the essential content of the present invention are still within the protection scope of the present invention.

[0069] Experimental examples and design of the method of this invention

[0070] This invention innovates and improves upon the following aspects through process exploration in the preparation of recombinant type A botulinum toxin.

[0071] First, regarding the design of the activation and enzymatic digestion process, the inventors discovered that traditional methods, such as those in patent CN120350044A, while not introducing exogenous enzyme cleavage sites during the preparation of recombinant botulinum toxin type A, suffer from slow digestion rates due to the low rate of enterokinase action. Furthermore, the digestion environment parameters are unfavorable to the stability of the activated toxin, and over-digestion can occur. The inventors addressed this by adjusting the type of enzyme, selecting trypsin for testing. Simultaneously, they utilized a method of binding the protein with purified packing material before enzymatic digestion for activation. By leveraging the physical steric hindrance formed by the binding of the cationic purified packing material to the toxin protein, they resolved the issue of over-cleavage at the C-terminus of the light chain.

[0072] Specifically, after activation by protease digestion, the natural amino acid sequence of botulinum toxin type A is cleaved from a single complete peptide chain into two peptide chains: a heavy chain (Hc) and a light chain (Lc), which are then linked by a pair of disulfide bonds. The cleavage sites are located between C432 and C448 at the C-terminus of the light chain (Lc), corresponding to the amino acid sequence RGIITSKTKSLDKGYNK. This peptide contains one arginine site and four lysine sites. When using trypsin to activate botulinum toxin, it is difficult to precisely cleave a single site; all five sites may be cleaved. To achieve precise cleavage, most traditional methods introduce specific cleavage sites at the cleavage sites, but this method can lead to inconsistencies between the toxin protein and the natural amino acid sequence.

[0073] The applicant's prediction of the pre-activation structure of botulinum toxin type A (e.g.) Figure 1 As shown in the figure, the circular region (indicated by the arrow) is the C-terminal region of the light chain (C430-C448), which corresponds to the region activated by enzyme cleavage. This region has a simple structure and is clearly exposed, making precise enzyme cleavage difficult in practice, and it is easy to cleave to C432. Considering that a certain region downstream of position 432 of the light chain is a region with a high concentration of positive charge, its average pI is greater than 9, the cation exchange chromatography packing can bind to this region (the theoretical pI of the fusion protein expression product of this invention is 5.69. Although the protein is negatively charged as a whole at pH 7.8-8.2, the downstream of the C-terminus of the light chain is a region with a high concentration of positive charge. The positive charge and exposure properties of this region mean that the protein can still bind to the cation exchange chromatography packing when the pH is greater than the pI value of the toxin protein). Therefore, it is speculated that steric hindrance can be generated after binding, thereby creating C-terminal protection of the light chain. That is, the steric hindrance after the cationic purification filler binds to the toxin can effectively protect the arginine at the C432 position from being cleaved by trypsin, so that the C-terminus of the light chain Lc is cleaved to the C438 position. That is, the C-terminal sequence of the cleaved light chain is RGIITSK, which is consistent with the C-terminal amino acid sequence of the light chain of Clostridium botulinum extract. When the C-terminus of the light chain is RGIITSK, the protein activity is significantly higher than that of the C432 cleaved type.

[0074] Conventional enzymatic digestion processes involve the reaction of proteases and toxin proteins in solution. After digestion, protease inhibitors are added, followed by purification to remove the protease and achieve true termination of the digestion reaction. However, the addition of protease inhibitors negatively impacts the product, and the purification conditions and duration significantly affect the activity of the toxin protein. This invention's enzymatic activation process, using on-column activation, ensures that the activated toxin protein remains bound to the cationic purification packing material after activation. It can be directly eluted with buffer solution. Trypsin and the cleaved MBP+HIS precursor tag do not bind to the packing material and flow rapidly through the column with the buffer, achieving rapid termination of digestion activation while simultaneously purifying the toxin protein.

[0075] Second, in the design of recombinant protein sequences

[0076] 1) In terms of label design, in order to ensure the correct folding and solubility of toxin proteins, this invention selects MBP as a solubilizing label. This label itself folds quickly and has extremely high solubility, which can promote the formation of soluble complexes of downstream proteins and significantly reduce the proportion of inclusion bodies.

[0077] In addition, since there may be undigested intact molecules remaining in the protein sample after enzyme digestion and activation, this invention also selects a 6×His tag as a purification tag to further remove these intact molecules; and in order to increase the exposure space and binding efficiency of the His tag, an artificial linker NNNNNNNNNNNGGGGS is introduced between the MBP tag and the His tag.

[0078] Therefore, the full sequence of the tag protein designed in this invention includes "MBP tag + adapter sequence + His tag", and its amino acid sequence is shown in SEQ ID NO.1:

[0079] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNGGGGSHHHHHH(SEQ ID NO.1)

[0080] The corresponding nucleic acid sequence is shown in SEQ ID NO.3:

[0081]

[0082] 2) Regarding the selection of flexible linkers, to effectively remove the tag sequence during the process, this invention also introduces a flexible linker between the tag and the toxin protein. The linker's sequence can be any combination of amino acids G and S. To ensure that no non-natural amino acid sequence remains at the N-terminus of the light chain after enzymatic digestion, amino acid K or R must be introduced at the C-terminus of the flexible linker, thereby ensuring that the trypsin can effectively remove both the tag protein and the flexible linker.

[0083] Therefore, the flexible connector sequence of the present invention is a combination of G and S, and the end of sequence C is R or K, such as GGGSR, GGGSK, GSSGSSR, GSSGSSK, GGSGGGSR or GGSGGGSK, etc.

[0084] 3) Regarding the selection of the BONT / A sequence, due to the special enzymatic digestion and activation process of this invention, the natural protein sequence can be directly selected for the BONT / A light / heavy chain during the recombinant expression process, without the need to introduce exogenous protease recognition sites between the light and heavy chains. Its amino acid sequence is shown in SEQ ID NO.2:

[0085] MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLT SIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAV TLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTS GKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK TKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL (SEQ ID NO.2)

[0086] The corresponding nucleic acid sequence is shown in SEQ ID NO.4:

[0087]

[0088] Based on the aforementioned design concept, this invention establishes a method for preparing recombinant type A botulinum toxin, the core steps of which include the following aspects:

[0089] 1) Construction of recombinant expression vector: The nucleic acid encoding the aforementioned tag sequence, flexible adapter sequence and BONT / A sequence is constructed into a nucleic acid construct and inserted into the expression vector to construct a recombinant expression vector; the construct is then transformed into a recombinant bacterium such as Escherichia coli to prepare recombinant bacteria; the recombinant bacteria are then fermented and cultured to express the toxin protein.

[0090] 2) Purification and activation of the toxin protein: The toxin protein undergoes a first purification treatment, an activation enzyme digestion treatment, a second purification treatment, and a third purification treatment to finally obtain the purified recombinant protein. Among them, the activation enzyme digestion involves binding the toxin protein with a cation exchange packing material, then using trypsin to activate the enzyme digestion treatment, and then rapidly terminating the activation enzyme digestion.

[0091] 3) Identification of recombinant protein: The activity of the purified recombinant protein was identified.

[0092] Example 1: Preparation of recombinant type A botulinum toxin

[0093] 1. Construction of recombinant bacteria and protein expression

[0094] The nucleic acid construct containing the aforementioned tag nucleic acid sequence (SEQ ID NO.3), flexible adapter (encoding the GGGSR nucleic acid sequence), and BONT / A nucleic acid sequence (SEQ ID NO.4) was ligated to the linearized expression vector pMal-C2X using a BM seamless ligation kit and transformed into E. coli BL21 competent cells. The transformation product was plated on solid LB agar plates containing 50 μg / ml ampicillin and incubated overnight at 37°C. Recombinant E. coli BL21 was cultured in LB medium at 37°C with shaking at 180-200 rpm for 8-12 hours, then inoculated into a fermenter and cultured at 37°C, pH 7.0, and dissolved oxygen 20%-70%, with appropriate feeding as needed based on bacterial growth. Once the bacterial OD600 was greater than 16, an appropriate amount of IPTG was added to induce expression at 18-22°C for 8-16 hours.

[0095] Collect the fermentation broth, centrifuge at 6000 rpm for 15 min to collect the bacterial cells, and resuspend in buffer. High-pressure homogenize the bacterial cells 2-3 times at 600-900 bar, and filter through a 0.22 μm sterile filter to obtain the toxin-derived protein extract.

[0096] 2. First purification by cation exchange chromatography of the toxin source

[0097] To initially purify the toxin-derived protein extract, the extract was first subjected to routine cation exchange chromatography purification, as detailed below:

[0098] Purification method: cation exchange chromatography.

[0099] The preferred packing material is SP Sepharose High Performance (Cytiva); Eluent A: 50 mM Tris-HCl pH 7.8; Eluent B: 50 mM Tris-HCl 1 M NaCl pH 7.8.

[0100] After the toxin-generating protein extract is adsorbed by the packing material, it is rinsed with elution buffer A and then eluted with 20% elution buffer B to obtain the target protein, thus obtaining the purified toxin-generating solution.

[0101] SDS-PAGE electrophoresis results are as follows: Figure 2 As shown, band 1 is the clarifying solution, band 2 is the permeation solution, band 3 is the elution solution, band 4 is the purified toxin protein from the first step, and band 5 is 100% solution B. It can be seen that the toxin protein (sequence including tag + light chain + heavy chain) has a molecular weight of approximately 180 kDa, and its purity is significantly improved after the first purification step.

[0102] 3. Activation and enzymatic digestion of trypsin

[0103] Place the protein separation and purification system, chromatography column, buffer solution and enzyme solution in a constant temperature chromatography cabinet and control the temperature at 20-25℃.

[0104] The cation exchange packing material used was SP Sepharose High Performance (Cytiva); Eluent A: 20 mM Tris-HCl pH 8.2; Eluent B: 20 ​​mM Tris-HCl 1 M NaCl pH 8.2.

[0105] Determine the column packing volume based on the total protein content of the toxin purification solution: pack more than 5 ml of packing material per 1 mg of toxin protein. Dilute the toxin purification solution with elution buffer A to a conductivity of 2-4, and load the sample at a flow rate of 3-6 ml / min. The toxin protein binds to the cationic packing material, and then elute with elution buffer A for 3-4 column volumes.

[0106] Based on trypsin activity, a 1 U / ml working solution of trypsin was prepared by diluting the sample with 20 mM Tris-HCl pH 8.2 buffer containing 2 mM CaCl2. The solution was fed at a flow rate of 1 ml / min for 1 hour. After digestion, the sample was washed with elution buffer A for 3 column volumes, followed by elution with 15% elution buffer B to obtain the activated protein. At this point, the sample had completed the removal of trypsin and the MBP+His tag, and the digestion was terminated.

[0107] Electrophoresis diagram of activation protein as shown Figure 3As shown in Figure a, the activation protein was detected by electrophoresis in both its non-reduced and reduced states. The non-reduced sample showed that trypsin could cleave the MBP+His tag of the toxinogen protein, and after rinsing with buffer, the eluted toxin protein was approximately 150 kDa. The reduced sample showed that trypsin cleaved the C-terminus of the light chain Lc, and the reduced component consisted of the heavy chain Hc (approximately 100 kDa) and the light chain Lc (approximately 50 kDa).

[0108] Simultaneously, using a traditional enzymatic digestion method, 1 U of trypsin was directly added to every 1 mg of toxinogen, and the mixture was incubated at pH 8.0 and 37°C for 30 minutes. After activation, the toxin protein was detected by protein electrophoresis. Figure 3 (b) Compared with the results obtained by the method of the present invention, the samples processed by the traditional method contain residual MBP+His tags (approximately 43 kDa), which require further purification for removal. Additionally, a stray band appears near 105 kDa in the non-reduced sample; this band disappears after reduction, indicating that it represents toxin protein resulting from excessive enzymatic digestion. Therefore, the method of the present invention saves at least one purification step.

[0109] 4. Second purification by affinity chromatography

[0110] Because trace amounts of intact protein molecules may remain in the sample after enzyme digestion and activation, this invention can further utilize the His tag on the intact molecule for purification and removal to ensure protein consistency, as detailed below:

[0111] Purification method: Ni affinity chromatography;

[0112] Eluent A: 20mM Tris-HCl, 150mM NaCl, pH 8.0; Eluent B: 20mM Tris-HCl, 150mM NaCl, 500mM imidazole, pH 8.0.

[0113] The activated protein was loaded onto a Ni column. Residual tagged molecules in the sample could bind to the Ni column. The flow-through was collected using elution buffer A to obtain the second purified solution. The column was then regenerated using elution buffer B. The second purification result is shown below. Figure 4 As shown.

[0114] 5. Third purification by molecular sieve chromatography

[0115] Given the presence of inherent toxin aggregates and small molecule impurities, and to facilitate the replacement of the protein buffer system, this invention preferably includes a third purification step using molecular sieving, as detailed below:

[0116] The preferred filler material is Superdex 200 prepgrade (Cytiva).

[0117] Mobile phase: 0.01M PB, 150mM NaCl, pH 7.4

[0118] The Superdex 200 prepgrade (Cytiva) was washed with 0.5 mol / L NaOH for two column volumes, followed by washing with purified water until the pH was neutral.

[0119] Wash the column with PBS, pH 7.4 solution for 3 cycles, and then load the second purification solution onto a Superdex 200 prepgrade (Cytiva) column at a flow rate of 30 cm / h, with a loading volume of 2%-5% each time.

[0120] Toxin aggregates and small molecule impurities were removed by molecular sieve chromatography, and the buffer system was replaced with PBS. The resulting stock solution (BONT / A stock solution) was obtained after 0.22 μm sterile filtration. The stock solution was then concentrated to obtain the final purified protein BONT / A. Electrophoresis analysis of the purified protein was performed. Figure 5 The results show that the sample has high purity and no visible impurities.

[0121] Example 2: N / C-terminal amino acid sequence analysis and detection

[0122] This embodiment evaluates the N / C ends of the recombinant purified protein prepared in Example 1 using liquid chromatography-mass spectrometry (LC-MS). The enzymatically digested samples are separated by UPLC, then identified by MS, and finally the mass numbers of the parent ions and secondary fragment ions of the protein and polypeptide are matched using BiopharmaFinder to confirm the amino acid linkage of the sequence.

[0123] Instrumentation and software: Ultra-high performance liquid chromatograph: Waters, I-Class Bio System; Column: AcquityUPLC CSH130C18 Column, Waters (1.7μm, 2.1*150mm); Mass spectrometer: Thermo QE-HFX; Xcalibur: Xcalibur 4.2; Bioharma Finder: BioharmaFinder 5.2.

[0124] Materials and reagents: Iodoacetamide (IAM) Sigma, Dithiothreitol (DTT) Bio-Rad, Trypsin enzyme Promega, Formic acid (FA) Fisher Scientific, Acetonitrile (ACN) Fisher, Water (H2O) Watsons, Ultrafiltration tubes Millipore, Centrifuge tubes Axygen

[0125] Experimental steps: 1) Denaturation and reduction: Take the purified protein prepared in Example 1, add Gdn-HCl and DTT, and perform denaturation and reduction treatment in a water bath at 37℃ for 30 min; 2) Alkylation: Add IAM to the denatured and reduced sample and place it in the dark for 30 min; 3) Enzyme digestion with liquid change: After liquid change, add Trypsin enzyme and digest overnight at 37℃; 4) Termination of reaction: Add an appropriate amount of FA solution to terminate the enzyme digestion reaction; 5) Sample loading and analysis: Use a water / acetonitrile mobile phase system, use a C18 column with surface charged hybrid particles for liquid phase separation, and use high-resolution mass spectrometry to detect the primary mass number of peptides and match the secondary fragment mass number of HCD to identify peptides.

[0126] Based on the above liquid chromatography-mass analysis, the C-terminal sequence of the light chain of the protein of this invention after Trypsin digestion was identified as GIITSK. Figure 6 ); the heavy chain C-terminal sequence is TLGCSWEFIPVDDGWGERPL ( Figure 7 Table 1 summarizes the C-terminal sequence identification results of the test samples.

[0127] Table 1. Summary of C-terminal sequence identification results

[0128] The N-terminal sequence of the light chain of the protein of this invention, after digestion with Trypsin, was identified as MPFVNK. Figure 8 ); the N-terminal sequence of the heavy chain is ALNDLCIK ( Figure 9 Table 2 summarizes the identification results of the N-terminal sequences of the light and heavy chains of the test samples.

[0129] Table 2. Summary of N-terminal sequence identification results

[0130] Yagi N-terminal sequence Mass-to-charge ratio m / z Number of charges Theoretical molecular weight Da Measured molecular weight (Da) Light chain MPFVNK 368.1951 2 734.3785 734.3745 Heavy chain ALNDLCIK 946.5001 1 945.4954 945.4923

[0131] Therefore, it can be seen that the recombinant purified protein BONT / A prepared by this invention has N / C terminal amino acid sequences in both the light and heavy chains that are consistent with theoretical values ​​and are consistent with natural Clostridium botulinum extract-based BONT / A products.

[0132] Simultaneously, this embodiment also performs N / C-terminal amino acid sequence analysis on the recombinant purified protein BONT / A obtained by the traditional enzymatic digestion method (i.e., adding 1U of trypsin to each 1mg of toxinogen, and reacting at pH 8.0 and 37℃ for 30 minutes).

[0133] The C-terminal sequence of the light chain of BONT / A protein prepared by conventional enzyme digestion method after Trypsin digestion was identified as LLCVR. Figure 10 ); the heavy chain C-terminal sequence is TLGCSWEFIPVDDGWGERPL ( Figure 11 Table 3 summarizes the results of C-terminal sequence identification of the test samples.

[0134] Table 3. Summary of C-terminal sequence identification results

[0135] The N-terminal sequence of the light chain of BONT / A protein prepared by conventional enzyme digestion method after Trypsin digestion was identified as MPFVNK. Figure 12 ); the N-terminal sequence of the heavy chain is ALNDLCIK ( Figure 13 Table 4 summarizes the identification results of the N-terminal sequences of the light and heavy chains of the test samples.

[0136] Table 4. Summary of N-terminal sequence identification results

[0137] Yagi N-terminal sequence Mass-to-charge ratio m / z Number of charges Theoretical molecular weight Da Measured molecular weight (Da) Light chain MPFVNK 368.1980 2 734.3785 734.3803 Heavy chain ALNDLCIK 473.7585 2 945.4954 945.5013

[0138] This shows that the recombinant purified protein BONT / A prepared by traditional enzymatic digestion process loses the GIITSK peptide at the C-terminus of the light chain, shortening the light chain amino acid count to 432 aa. Meanwhile, the N / C-terminus of the heavy chain remains intact, resulting in better stability.

[0139] Example 3: Evaluation of isoelectric point using capillary isoelectric focusing electrophoresis

[0140] In this embodiment, the isoelectric point of the recombinant purified protein BONT / A prepared in Example 1 was evaluated using capillary isoelectric focusing electrophoresis.

[0141] Laboratory instruments and reagents / consumables:

[0142]

[0143] 35 μL of 1% methylcellulose, 4 μL of 3-10 amphoteric electrolyte, 2 μL of 0.5 M arginine, 1 μL per pI Marker, 5 μL of the recombinant purified protein prepared in Example 1, and 52 μL of ultrapure water were mixed into a 1.5 ml centrifuge tube and vortexed. 95 μL of the sample was transferred to a 96-well plate and placed in the sample tray of the instrument for analysis. A blank control without sample was also included.

[0144] The instrument parameters are set as follows:

[0145] name content Focus Period 1 1500V for 1.0min Focus Period 2 3000V for 6.0min Sample Load Duration 55.0Seconds pImarker1 4.1 pImarker2 9.5 Tray Temperature 10.0±2℃

[0146] Specific test results are as follows: Figure 14 As shown, the measured isoelectric point peak of the protein of this invention in this embodiment is 6.283, which is comparable to the theoretical value of pI 5.9.

[0147] Example 4: Protein Purity Detection

[0148] In this embodiment, SEC-MALS was used to detect the recombinant protein BONT / A prepared in this invention.

[0149] Experimental materials included: Shimadzu HPLC (model Lc-20AD), Shimadzu RID-20A differential refractive index detector, Shimadzu SPD-20A UV-Vis detector; chromatographic column: NanoChrom BioCoreSEC-300, 5μm 7.8×300mm; mobile phase: 0.1mol / L PBS, pH 7.0; flow rate: 0.5ml / min; column temperature: 30℃; run time: 40min.

[0150] Test results as follows Figure 15 As shown in Table 5.

[0151] Table 5. Purity test results of the recombinant stock solution BONT / A of the present invention

[0152]

[0153] As can be seen, the recombinant purified protein prepared by the method of the present invention can achieve a protein purity of 98.792%.

[0154] Example 4: Determination of protein toxicity and specific activity

[0155] In this embodiment, the recombinant protein prepared according to the Chinese Pharmacopoeia standard was diluted according to the dilution ratios in Table 4. Subsequently, each dilution was injected intraperitoneally into 26-30 day old SPF-grade Kunming mice, with each mouse receiving 0.5 mL. The mortality rate of the animals within 4 days was statistically analyzed, and the results are shown in Table 6.

[0156] Table 6. Results of toxicity and specific activity assays of the recombinant protein of this invention

[0157]

[0158]

[0159] The Reed-Muench method was used to calculate protein toxicity and specific activity, as follows:

[0160] Distance ratio: (85.71-50) / (85.71-33.33)=0.7

[0161] Logarithm of 50% mortality endpoint = Logarithm of dilution factor above 50% mortality rate + Logarithm of distance ratio × dilution factor = 7.2 + 0.7 × 0.301 = 7.41

[0162] Dilution: 10 7.41

[0163] Poison power: 10 7.41 ×2=5.14×10 7 LD 50 / ml

[0164] Specific activity: Toxicity per mg of toxin = 5.14 × 10⁻⁶ 7 LD 50 / ml ÷ 0.102mg / ml = 5.03 × 10 8 LD 50 / mg.

[0165] Meanwhile, the toxicity and specific activity of the stock solution obtained by the traditional enzymatic digestion method (i.e., adding 1U of trypsin to each 1mg of toxinogen and reacting at pH 8.0 and 37°C for 30 minutes) were also tested. The specific results are shown in Table 7 below.

[0166] Table 7. Results of BONT / A toxicity and specific activity obtained by traditional enzyme digestion methods

[0167]

[0168] Using the same statistical methods as above, the toxicity was determined to be: 10. 6.39 ×2=2.45×10 6 LD 50 / ml, specific activity: toxicity per mg of toxin = 2.45 × 10 6 LD 50 / ml ÷ 0.102mg / ml = 2.41 × 10 7 LD 50 / mg.

[0169] Comparative analysis shows that the recombinant purified protein prepared by this invention has improved toxicity and specific activity by about 20 times compared with traditional methods.

[0170] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A BONT / A nucleic acid construct, characterized in that, The nucleic acid construct comprises a tag sequence and a BONT / A nucleic acid sequence; the tag sequence encodes an MBP tag and a His tag; the BONT / A nucleic acid sequence does not introduce a restriction site.

2. The BONT / A nucleic acid construct of claim 1, wherein, The tag sequence encodes an MBP tag, an artificial linker sequence and a His tag in sequence. Preferably, the artificial linker sequence is NNNNNNNNNNGGGGS. More preferably, the tag sequence is a nucleic acid sequence encoding SEQ ID NO.

1.

3. The BONT / A nucleic acid construct according to any one of claims 1-2, wherein, The BONT / A nucleic acid sequence encodes a native BONT / A protein sequence. Preferably, the native BONT / A protein sequence is shown in SEQ ID NO.

2.

4. The BONT / A nucleic acid construct according to any one of claims 1 to 3, wherein, The BONT / A nucleic acid construct further comprises a flexible linker sequence, which connects the tag sequence and the BONT / A nucleic acid sequence. Preferably, the flexible linker sequence encodes an amino acid linker, which is any combination of G and S, and the sequence C-terminal end is R or K. More preferably, the amino acid linker is selected from the following sequences: GGGSR, GGGSK, GSSGSSR, GSSGSSK, GGSGGGSR or GGSGGGSK.

5. A method for preparing a recombinant botulinum toxin type A, characterized by, The method comprises the following steps: 1) Recombinant protein expression: the BONT / A nucleic acid construct of any one of claims 1-4 is subjected to recombinant expression under conditions suitable for protein expression; 2) First purification of recombinant protein: the expression product is purified to obtain a first purified product; 3) Activation of activated protein by protease cleavage: the first purified product is combined with a purification filler, and activated by trypsin activation treatment; after activation, the activated protein is obtained by elution according to the elution mode suitable for the purification filler.

6. The preparation method according to claim 5, characterized in that, The conditions suitable for protein expression in step 1) include: inserting the BONT / A nucleic acid construct of any one of claims 1-4 into a plasmid to construct a recombinant expression vector, constructing the recombinant expression vector into a recombinant bacterium, and performing fermentation culture and protein expression on the recombinant bacterium.

7. The method of any one of claims 5-6, wherein, In step 2), the purification comprises cation exchange chromatography purification.

8. The method of any one of claims 5-7, wherein, In step 3), the purification filler is a cation exchange chromatography filler; and the use form of the purification filler is selected from a chromatography column form or a filler suspension form.

9. The method of any one of claims 5-8, wherein, The preparation method further comprises a second purification and / or a third purification; preferably, the second purification is Ni column affinity purification, and the third purification is molecular sieve purification.

10. A recombinant botulinum toxin of type A, characterized in that The recombinant botulinum toxin type A is prepared by the method of any one of claims 5-9.

Images