Botulinum toxin a1 precursor molecule, nucleic acid molecule, engineered bacteria and preparation method thereof, and method for preparing natural botulinum toxin a using the precursor molecule

Abstract

This invention relates to a botulinum toxin A1 precursor molecule, a nucleic acid molecule, an engineered bacterium, a method for preparing the same, and a method for preparing natural botulinum toxin A using the precursor molecule. The precursor molecule comprises a light chain and a heavy chain, and a linker region connecting the light and heavy chains. The amino acid sequence of the precursor molecule is shown in SEQ ID NO. 4. The light chain is the amino acid sequence from position 1 to position 444 of natural botulinum toxin A, as shown in SEQ ID NO. 5. The heavy chain sequence is the amino acid sequence from position 449 to position 1296 of natural botulinum toxin A, as shown in SEQ ID NO. 6. The amino acid sequence of the linker region is shown in SEQ ID NO. 11. This invention provides a safe process, and the resulting recombinant botulinum toxin not only has a sequence identical to the natural botulinum toxin active protein but also exhibits high activity, providing a low-cost, high-activity botulinum toxin product for clinical use.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to botulinum toxin A1 precursor molecules, nucleic acid molecules, engineered bacteria, and preparation methods thereof for the preparation of natural botulinum toxin A, as well as methods for preparing natural botulinum toxin A using the precursor molecules. Background Technology

[0002] Botulinum neurotoxins (BoNTs), commonly known as botulinum toxin, are neurotoxins produced by Clostridium botulinum. They possess potent neurotoxicity, being 600 million times more toxic than sodium cyanide and 30 million times more toxic than cobra venom. Based on serotype, botulinum toxin can be classified into seven types, from A to G. Currently, the vast majority of commercially available drugs are type A, such as natural botulinum toxin A, whose amino acid sequence is shown in SEQ ID NO. 7.

[0003] Type A botulinum toxin, in its natural state, is a complex of approximately 900 kDa composed of multiple proteins. The core is a 150 kDa toxin protein, while the remaining proteins do not exhibit toxicity and primarily protect the toxin protein from the low pH and various digestive enzymes in the digestive system. Most commercially available botulinum toxins are purified from the 900 kDa complex. A few products, such as Xeomin from Merz (Germany), have removed the accessory proteins, retaining only the 150 kDa core protein. This reduces the risk of immune responses and antibody production, and avoids the gradual decline in efficacy caused by repeated administration.

[0004] The world's first commercially available botulinum toxin product was Botox, manufactured by Allergan in the United States. Approved by the FDA in 1989, it was used to temporarily improve moderate to severe frown lines caused by activity of the corrugator supercilii and / or depressor supercilii muscles in adults aged 65 and under. Botox, and most subsequent commercially available botulinum toxin products, including the only approved domestically produced botulinum toxin, Hengli, are derived from the fermentation of Clostridium botulinum Hall strain. The production process requires a very strict anaerobic environment and is temperature-sensitive. Combined with the extremely high toxicity of botulinum toxin itself, the control of the fermentation and purification processes is extremely stringent. Therefore, there is a need to develop a botulinum toxin preparation method that does not exhibit extremely high toxicity in the early stages of fermentation and purification, which would greatly benefit the industry's development.

[0005] Escherichia coli is one of the most widely used expression systems in the field of recombinant proteins, with advantages such as clear genetic background, simple culture operation, high transformation and transduction efficiency, rapid growth and reproduction, and low cost. However, its disadvantages include a weak ability to form disulfide bonds and the lack of post-translational modification. Although botulinum toxin itself has a relatively large molecular weight, it only has two pairs of disulfide bonds and does not undergo post-translational modification, making Escherichia coli a suitable expression system.

[0006] The 150kD core protein of botulinum toxin consists of two subunits, 50kD and 100kD. It is synthesized in its complete precursor protein form, which is inactive. Then, a protease cleaves several amino acids between the two subunits to form the active form. The 50kD subunit is the light chain (LC), and the 100kD subunit is the heavy chain (HC), linked by a disulfide bond. HC specifically binds to presynaptic membrane receptors to target nerve cells, and then the disulfide bond is broken, allowing the LC to enter the nerve cell. LC exhibits Zn-dependent activity. 2+ Due to its metalloproteinase activity, botulinum toxin type A can specifically degrade the SNAP25 protein, a signaling molecule in cells that transmits nerve impulses, thereby blocking neuromuscular transmission.

[0007] Several strategies exist for the expression of recombinant botulinum toxin. Chinese patent CN115785237A constructs HC and LC separately within two reading frames of an expression vector, achieving co-expression in *E. coli* and intracellular assembly into active botulinum toxin. While simple, this method fails to address the issue of safe botulinum toxin production because active botulinum toxin is generated during fermentation. Another Chinese patent CN114989271A expresses LC and HC separately, then denatures and refolds them together in vitro. During refolding, some HC and LC can assemble into active botulinum toxin, but the efficiency is low, only about 30%. Furthermore, multiple purification steps are required after refolding, increasing the possibility of contamination. In conclusion, expressing inactive full-length botulinum toxin precursor protein in *E. coli*, purifying it to a high purity, and then cleaving the linker peptide between LC and HC using a specific protease to activate botulinum toxin activity represents a relatively safe production method.

[0008] Several patents currently involve this method. Chinese patent CN114957482A uses a 3C protease as the cleavage enzyme and fuses a GST tag to the N-terminus of the protein. A 3C cleavage site is also added between the GST tag and the botulinum toxin. While activating the protein with the 3C enzyme can remove the tag, several amino acids remain at both ends of the LC and the N-terminus of the HC. Another Chinese patent, CN 117925581A, studies more tags and cleavage sites, including GST, MBP, and His tags. The cleavage sites used include 3C protease, TEV protease, thrombin, and enterokinase. Tags are fused to both ends of the entire length of the botulinum toxin. This results in more amino acid residues after enzyme activation than the previous patent. In addition, Chinese patents with publication numbers CN118126143 A and CN118006523 A are similar, all containing amino acid residues. These residual amino acids may cause additional immune responses, and according to the patent report, their LD50 is about 0.5 ng / kg, which is lower than the reported natural protein activity of about 0.25–0.45 ng / kg (Toxins 2019, 11, 686).

[0009] Therefore, it is necessary to establish an expression sequence that is completely consistent with the natural sequence after cleavage and activation to obtain a highly active botulinum toxin product. Summary of the Invention

[0010] To address the above problems, this invention constructs a recombinant genetically engineered bacterium expressing type A botulinum toxin using genetic engineering technology, aiming to obtain a recombinant botulinum toxin with a natural sequence and high activity.

[0011] According to a first aspect of the present invention, the present invention provides a botulinum toxin A1 precursor molecule for preparing natural botulinum toxin A, the botulinum toxin A1 precursor molecule comprising a light chain and a heavy chain, and a linker region connecting the light chain and the heavy chain, the amino acid sequence of the botulinum toxin A1 precursor molecule being shown in SEQ ID NO. 4; wherein, the light chain (LC) is the amino acid sequence from position 1 to position 444 of natural botulinum toxin A, the amino acid sequence of which is shown in SEQ ID NO. 5, the heavy chain (HC) sequence is the amino acid sequence from position 449 to position 1296 of natural botulinum toxin A, the amino acid sequence of which is shown in SEQ ID NO. 6, the amino acid sequence of the linker region (LLH) being shown in SEQ ID NO. 11, and the amino acid sequence of natural botulinum toxin A being shown in SEQ ID NO. 7.

[0012] In the botulinum toxin A1 precursor molecule of the present invention, preferably, the botulinum toxin A1 precursor molecule includes two protease Kex2 cleavage sites, the two protease Kex2 cleavage sites being located at KR at positions 444 and 445, and KR at positions 455 and 456 of the amino acid sequence shown in SEQ ID NO. 4, respectively.

[0013] Here, the inventors experimentally discovered that introducing a 6×His(HHHHHH) sequence into the sequence facilitates purification; introducing two Kex2 restriction sites into the sequence facilitates tag removal, protein activation, and protein purification. The resulting nucleic acid fragment was then assembled and inserted into a prokaryotic expression vector.

[0014] It should be noted that, given the homology of sequence structures among various serum botulinum toxin types, the natural botulinum toxin protein covered by this invention is selected from any one of type A, B, C1, C2, D, E, F, G, or H botulinum toxin proteins. Preferably, the natural botulinum toxin protein is natural botulinum toxin A protein with the amino acid sequence shown in SEQ ID NO.7.

[0015] According to a second aspect of the present invention, the present invention provides a nucleic acid molecule encoding the botulinum toxin A1 precursor molecule, the nucleic acid molecule being shown in SEQ ID NO.3, wherein the nucleic acid molecule encoding the light chain is shown in SEQ ID NO.8, the nucleic acid molecule encoding the heavy chain is shown in SEQ ID NO.9, and the nucleic acid molecule encoding the linker region is shown in SEQ ID NO.10.

[0016] According to a third aspect of the present invention, the present invention provides an engineered bacterium for producing the botulinum toxin A1 precursor molecule, the engineered bacterium containing the nucleic acid molecule.

[0017] In the engineered bacteria for the botulinum toxin A1 precursor molecule described in this invention, preferably, the engineered bacteria is *Escherichia coli*. The constructed recombinant vector is transferred into an *E. coli* expression strain to construct a genetically engineered bacterium expressing the target protein.

[0018] According to a fourth aspect of the present invention, the present invention provides a method for preparing the botulinum toxin A1 precursor molecule, the method comprising the following steps:

[0019] 1) The 000C(KK) amino acid sequence shown in SEQ ID NO.2 was inserted into a prokaryotic expression vector to obtain the 000C(KK) vector;

[0020] 2) Using overlap extension PCR, all KK sites on the 000C(KK) amino acid sequence were mutated to KR, resulting in RHHHHHHGYNKR at positions 445-456 of the 000C(KK) amino acid sequence, and the KR sites at both ends of the 6×His tag having two Kex2 restriction sites, to obtain the mutated sequence; and

[0021] 3) The mutated sequence is ligated to the 000C(KK) vector to obtain a recombinant vector with the Top10-000C(2KR) sequence, the amino acid sequence of which is shown in SEQ ID NO.4.

[0022] In the method for preparing botulinum toxin A1 precursor molecules according to the present invention, preferably, the prokaryotic expression vector is pET28, pET32, pQE30, pQE60, or pGEX-6P.

[0023] According to a fifth aspect of the present invention, the present invention provides a method for preparing natural botulinum toxin A using the botulinum toxin A1 precursor molecule, the method comprising the following steps:

[0024] 1) Transform the recombinant vector containing the amino acid sequence Top10-000C(2KR) shown in SEQ ID NO.4 into competent Escherichia coli cells to construct a genetically engineered bacterium expressing the target protein;

[0025] 2) The target protein is expressed by fermentation culture of the genetically engineered bacteria, and after purification, botulinum toxin precursor protein is obtained; and

[0026] 3) The botulinum toxin precursor protein was cleaved by Kex2 protease to remove the 6XHis tag and activate it simultaneously. Then, the excess arginine residue at the C-terminus of the light chain was removed by carboxypeptidase B to obtain natural botulinum toxin A as shown in SEQ ID NO.7.

[0027] In the method for preparing natural botulinum toxin A according to the present invention, preferably, in step 1), the Escherichia coli is BL21, BL21(DE3), BL21(DE3)plysS, BLR, BLR(DE3), BLR(DE3)pLysS, Rosetta(DE3), Origami B(DE3), Origami 2(DE3), Rosetta gami(DE3)pLysS, Rosetta gamiB(DE3), Rosetta gami 2(DE3), Shuffle T7-K12, Shuffle T7-B, or BL21 Star(DE3).

[0028] In the preparation method of natural botulinum toxin A described in this invention, after the constructed genetically engineered bacteria are fermented in a culture medium with optimized conditions to express the target protein, this invention establishes a complete set of purification methods, including but not limited to Ni column affinity chromatography, ion exchange chromatography, hydrophobic chromatography, and molecular sieves. In the preferred embodiment of this invention, the purification process preferably includes Ni column affinity chromatography, UniGel 30Q anion exchange chromatography, Q anion exchange chromatography, and SP cation exchange chromatography. The purified botulinum toxin precursor protein is obtained.

[0029] In the method for preparing natural botulinum toxin A according to the present invention, the purified botulinum toxin precursor protein is digested with Kex2 protease to remove the 6×His tag and simultaneously activate it. Then, excess arginine is removed using carboxypeptidase B to obtain the target product of the present invention. Preferably, in step 3), the Kex2 protease further includes a Kex2 digestion buffer, which is 50 mM Tris-HCl, pH 8.0, 0.1–2 mM CaCl2; or 50 mM Tris-HCl, pH 8.0, 0.1–2 mM CaCl2, 0.01%–0.5% Tween-20.

[0030] Experiments have shown that since botulinum toxin type A is cleaved after the two lysine residues (K) of the intermediate linker peptide during activation, this invention cleverly utilizes its own amino acid sequence to introduce arginine (R) after the lysine residues. The KR sequence is precisely the cleavage site of the protease Kex2. After Kex2 cleavage, no amino acids remain at the N-terminus of HC, but arginine remains at the C-terminus of LC. Then, by using carboxypeptidase B to remove the excess arginine, botulinum toxin type A with a completely identical sequence to the natural one can be obtained.

[0031] In addition, the tool enzymes used (such as KEX2 enzyme, carboxypeptidase B, etc.) can be removed and further purified by methods such as Ni column affinity chromatography and ion exchange chromatography to obtain the final product.

[0032] The beneficial effects of this invention are as follows: 1) Before activation by the tool enzyme, the target protein is in an inactive precursor protein state, making production safer; 2) After activation, only 1 to 2 purification steps are needed to obtain the final product, thereby minimizing the possibility of contamination; 3) The recombinant botulinum toxin obtained by this invention is not only completely identical to the sequence of the natural botulinum toxin active protein, but also has high activity, thus providing a low-cost, high-activity, and more stable botulinum toxin product for clinical use; 4) Compared with the shortcomings of the existing technical routes for preparing botulinum toxin, this invention provides an environmentally friendly technical route.

[0033] It should be noted that the carriers, cells, enzymes, etc. mentioned in the embodiments are for the purpose of illustrating the invention, rather than limiting the scope of protection of the invention. Attached Figure Description

[0034] Figure 1 Agarose gel electrophoresis image of the recombinant vector pET28a-000C(2KR);

[0035] Figure 2a Sequencing results for the LC and HC linker regions in Top10-000C(2KR); Figure 2b , Figure 2c , Figure 2d , Figure 2e , Figure 2f , Figure 2g Sequencing results for the full-length coding region of Top10-000C (2KR);

[0036] Figure 3 SDS-PAGE electrophoresis image of purified 000C(2KR);

[0037] Figure 4 SDS-PAGE electrophoresis image of purified 000C(2KR) after concentration using an ultrafiltration tube;

[0038] Figure 5 SDS-PAGE electrophoresis image of 000C(2KR) purified by Kex2 digestion and magnetic beads;

[0039] Figure 6 Western blot image of 000C(2KR) purified by Kex2 digestion and magnetic beads;

[0040] Figure 7 SDS-PAGE electrophoresis image of SNAP25 after activation at 000C (2KR);

[0041] Figure 8 SEC detection chromatogram for 000C (2KR);

[0042] Figure 9 Peptide sequence analysis (LC) of 000C(2KR) after digestion with Kex2 and carboxypeptidase B;

[0043] Figure 10 Peptide sequence analysis (HC) of 000C (2KR) after digestion with Kex2 and carboxypeptidase B. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention.

[0045] The strains, plasmids, and reagents used in the embodiments of this invention are all commercially available products.

[0046] pET28a vector (Sigma, pET-28a(+)DNA-Novagen, model: 69864)

[0047] SalI-HF (NEB (New England Biolabs), model: 10206407);

[0048] EcoRI-HF (NEB (New England Biolabs), model: 10168528);

[0049] T4 ligase (NEB (New England Biolabs), model: 10201191);

[0050] Kanamycin (Shanghai Yisheng Biotechnology Co., Ltd., Model: K8325110);

[0051] Omega Plasmid Mini-Prep Kit (Omega, Model: D6943020000C22X046)

[0052] Escherichia coli BLR(DE3) (Beijing Zhuangmeng International Biotechnology Co., Ltd., Model: ZC1240-1);

[0053] IPTG (Beijing Solarbio Technology Co., Ltd., Model: 1213Z052);

[0054] Ni Trap-HP column (Cytiva (Cytiva Life Sciences), model: 10347449);

[0055] Gradient pre-cast adhesive (ACEbiotech., model: J73354102X);

[0056] Coomassie Brilliant Blue (Beyotime Biotechnology Co., Ltd., Model: Z968240829);

[0057] Pierce BCA Protein Quantitative Kit (Thermofisher Scientific, Model: ZB395549);

[0058] Kex2 protease (Shanghai Yaxin Biotechnology Co., Ltd., model: Re230301);

[0059] Kex2 protease (Aimeidi Biotechnology Co., Ltd., model: IM02P02(BP));

[0060] Carboxypeptidase B (MCE MedChemExpress, model: 323706);

[0061] Anti-His antibody (Proteintech, model: 21012256);

[0062] SNAP25 (Beijing Anruit Biotechnology Co., Ltd., Model: BARTSN01);

[0063] Gelatin (Aladdin, model: G108396-500g).

[0064] Example 1: Construction of Recombinant Vector

[0065] The 000C(KK) sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd. (wherein, the 000C(KK) sequence is a codon-optimized sequence, its nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2). The Xba I and Xho I sites of the pET28a vector were inserted into the 000C(KK) sequence. Both KK sites on the HC were mutated to KR using overlap extension PCR, so that the His tag has two Kex2 restriction sites at both ends. Here, the two Kex2 restriction sites are located at KR positions 444 and 445, and KR positions 455 and 456, respectively, in the amino acid sequence shown in SEQ ID NO.4.

[0066] The mutated sequence was ligated with the 000C(KK) vector, digested with EcoRI and SalI, and then ligated using T4 ligase. 5 μL of the ligation product was used to transform 50 μL of GenScript Top10 competent cells according to the manufacturer's instructions. The cells were then plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. Once colonies had grown, single colonies were picked and subjected to culture PCR for verification. The results are as follows... Figure 1 As shown, Figure 1 Agarose gel electrophoresis image of the PCR results for the recombinant vector pET28a-000C(2KR).

[0067] Positive clones were selected for Sanger sequencing. After verifying the sequence was correct, it was named Top10-000C(2KR). The nucleic acid sequence of the verified Top10-000C(2KR) is shown in SEQ ID NO.3, and its corresponding amino acid sequence is shown in SEQ ID NO.4. The sequence was sent to Qingke Biotechnology Co., Ltd. for sequencing, and the sequencing results were consistent with the design. See [link to results]. Figure 2a , Figure 2b , Figure 2c , Figure 2d , Figure 2e , Figure 2f , Figure 2g ,in, Figure 2a Sequencing results for the LC and HC linker regions in Top10-000C(2KR); Figure 2b , Figure 2c , Figure 2d , Figure 2e , Figure 2f , Figure 2g The sequencing results are for the full-length coding region of Top10-000C(2KR).

[0068] Example 2: Transformation of Escherichia coli expression strains

[0069] The correctly sequenced Top10-000C(2KR) strain was inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 250 rpm for 16–18 h. Plasmids were extracted using the Omega Plasmid Mini-Prep Kit (catalog number D6943-02) according to the manufacturer's instructions. 1 μL of plasmid was transformed into 50 μL of *E. coli* BLR(DE3) competent cells according to the method described in Molecular Cloning Laboratory Guide. The transformed plasmid was plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. Several clones were picked and inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C with shaking for colony PCR verification. One tube of correctly verified bacterial culture was named BLR(DE3)-000C(2KR). 8% sterile glycerol was added, and the culture was stored at -80°C.

[0070] Example 3: Expression of the target protein

[0071] Take 1 μL of bacterial culture from a BLR(DE3)-000C(2KR) cryovial and inoculate it into 3 mL of TB medium containing 50 μg / mL kanamycin. Incubate at 37°C with shaking for 16 h. Then inoculate into 10 mL of TB medium containing 50 μg / mL kanamycin to allow the initial OD to rise. 600 =0.02, incubate at 37℃ with shaking until OD 600 ≈1.0, inoculate at 1:100 into 1-2 L of TB medium containing 50 μg / mL kanamycin, and incubate at 37°C with shaking until OD. 600 When the concentration reaches approximately 0.6, filter-sterilized IPTG is added to a final concentration of 0.5 mM. The mixture is then incubated at 20°C with shaking at 250 rpm for 16 hours. The bacterial culture is collected, and the bacterial cells are collected by centrifugation at 4000 rpm for 10 minutes.

[0072] Example 4: Purification of the target protein

[0073] 1) Ni column chromatography process parameters:

[0074] Ni Trap-HP column

[0075] A. Equilibrium solution: 20mM Tris + 2M NaCl + 10mM ZnCl2 + 10% glycerol, pH 8.0

[0076] B. Sample preparation: The bacterial cells were resuspended in equilibration buffer at a ratio of 1:20 (w:v), lysed at 800 bar, centrifuged at 15000g for 15 min, and the supernatant was filtered and loaded onto the sample. The retention time was greater than 2 min.

[0077] C. Eluent: 20mM Tris + 2M NaCl + 10mM ZnCl2 + 10% glycerol, pH 8.0

[0078] 20mM Tris + 0.1M NaCl + 10mM ZnCl2 + 10% glycerol, pH 8.0

[0079] D. Eluent: 20mM Tris + 0.1M NaCl + 10mM ZnCl2 + 0.5M imidazole + 10% glycerol, pH 8.5

[0080] E. Cleaning: Rinse with 6M guanidine hydrochloride for 5-10 CV, then rinse with 2M NaCl for 5-10 CV, rinse with water, and store in 20% EtOH.

[0081] This method is applicable to E. coli samples expressing 000C (2KR).

[0082] 2) Q-anion chromatography:

[0083] A. Equilibrium solution: 20 mM Tris + 10% glycerol, pH 8.0 ± 0.2

[0084] B. Sample preparation: Elute the sample with a Ni column, dilute it with equilibration buffer, and then load the sample.

[0085] C. Eluent: 20mM Tris + 0.25M NaCl + 10% glycerol, pH 8.0±0.2

[0086] D. Cleaning: Rinse with 0.5M NaOH + 2M NaCl for 5CV, then rinse with water and store in 20% ethanol.

[0087] SDS-PAGE electrophoresis was used to determine the molecular weight of proteins. A 4-12% gradient precast gel (ACE) was used for detection at 160V for approximately 45-50 minutes. After electrophoresis, the proteins were stained with Coomassie Brilliant Blue rapid staining solution. The electrophoresis results are shown below. Figure 3 As shown, a clear band is visible at the 150kD position, indicating successful protein purification. The protein was concentrated approximately 70-fold using a 100kD ultrafiltration tube, and then analyzed by SDS-PAGE. The electrophoresis results are as follows. Figure 4 As shown.

[0088] Protein concentration was determined using the BCA method: The Pierce BCA protein quantification kit (Thermofisher) was used to determine protein concentration according to the manufacturer's instructions. Protein concentrations ranged from 0.1 to 3 mg / ml.

[0089] Protein purity was determined by high performance liquid chromatography (SEC) (see results). Figure 8 Column type: BioCore SEC-300 3μm, 4.6*300mm, Nanospectrum Analysis Technology (Suzhou) Co., Ltd.; Column temperature: 30℃; Detector: 280nm; Mobile phase composition: 90% 50mM phosphate buffer (pH 6.8, 300mM NaCl), 10% isopropanol; Flow rate: 0.35mL / min; Injection volume: 10μL; Protein purity can reach over 95%.

[0090] Example 5: Kex2 enzyme digestion of target protein and purification with magnetic beads

[0091] Add Kex2 digestion buffer (final concentration 50mM Tris-HCl, pH 8.0, 2mM CaCl2) to the purified protein, and add Kex2 protease (KEX2: botulinum toxin) at a mass ratio of 1:250-1000. Incubate at 25°C for 10-40 minutes to complete activation. Then add carboxypeptidase B (carboxypeptidase B: botulinum toxin) at a mass ratio of 1:335-1000, and incubate at 37°C for 10-30 minutes. Add equilibrated Ni magnetic beads to the digested protein, incubate on a rotary mixer for 0.5-1 hour, and adsorb the magnetic beads onto a magnetic rack. The supernatant is the de-labeled double-stranded protein sample, which is the target product of this invention.

[0092] Samples were taken for SDS-PAGE and Western Blot analysis. The amino acid sequence of the cleaved LC protein is shown in SEQ ID NO. 5, and the amino acid sequence of the cleaved HC protein is shown in SEQ ID NO. 6. Samples were sent to a testing company (Beijing Biotech Biotechnology Co., Ltd.) for mass spectrometry sequencing.

[0093] Mass spectrometry peptide mapping analysis showed that the test results were consistent with the theoretical calculations. The results are as follows: Figure 9 , Figure 10 As shown, Figure 9 Peptide sequence analysis (LC) of 000C(2KR) after digestion with Kex2 and carboxypeptidase B; Figure 10 Peptide sequence analysis (HC) of 000C (2KR) after digestion with Kex2 and carboxypeptidase B.

[0094] The methods for determining protein concentration and purity are the same as in Example 4. The results of the protein concentration and purity determination are as follows: protein concentration is 0.1-1 mg / L, and protein purity is 95%.

[0095] The SDS-PAGE detection method is the same as in Implementation Example 4, and the results are as follows: Figure 5 As shown, by Figure 5 It can be seen that the target protein is present in both the transudate and the elution. The transudate contains the protein that has been cleaved at both Kex2 sites, while the elution contains the protein that has not been completely cleaved and still contains the 6×His sequence, so it can be adsorbed by the magnetic beads.

[0096] After SDS-PGAE electrophoresis, proteins were transferred to a PVDF membrane using a semi-dry transfer method at 10V for 10 min, 25V for 30 min. Detection was then performed using an Anti-His antibody, and the results are as follows. Figure 6 As shown, by Figure 6 It can be seen that only when obvious bands are detected in the elution buffer does it indicate that the target protein in the flow-through solution has been completely cleaved.

[0097] Example 6: Validation of SNAP25 cleavage activity in vitro

[0098] Take purified SNAP25 and purified 000C (2KR), add SNAP25 digestion buffer (final concentration 50 mM HEPES, pH 7.5, 1% Tween-20, 50 mM ZnCl2, 25 mM DTT). Incubate at 37℃ for 30 min. Use undigested SNAP25 as a control, and take samples for SDS-PAGE analysis.

[0099] The SDS-PAGE detection method is the same as in Example 4, and the results are as follows: Figure 7 As shown, by Figure 7 As can be seen, the Snap protein was completely digested, and its size was consistent with the expected cleavage of the last 9 amino acids, indicating that the purified protein has the activity to cleave SNAP25, which is in line with expectations.

[0100] Example 7: Animal experiments to verify the in vivo activity of 000C (2KR) protein

[0101] Using gelatin phosphate buffer (2.0 g gelatin, 4.0 g disodium hydrogen phosphate (Na2HPO4), 1000.0 mL distilled water, pH adjusted to 6.2, autoclaved at 121°C for 15 min), undigested 000C(2KR) protein (hereinafter referred to as the undigested group) and Kex2-digested and purified 000C(2KR) protein (hereinafter referred to as the digested group) were serially diluted. Forty-two female Kunming mice were randomly divided into seven groups. After acclimatization for 3 days, 0.5 mL of each protein concentration was administered intraperitoneally to six mice in each group. Mice in the undigested group received 15.3 ng, 10.2 ng, 6.8 ng, 4.5 ng, 3 ng, 2 ng, and 1.3 ng of the drug, respectively. Mice in the digested group received 8 pg, 5.3 pg, 3.5 pg, 2.3 pg, 1.5 pg, 1 pg, and 0.7 pg of the drug, respectively. After administration, mice were fed for 4 days, and the number of deaths and viability were recorded.

[0102] Calculations showed that the LD50 of the enzyme-digested group was 3.1 pg, the LD50 of naturally extracted botulinum toxin was 20-60 pg, and the LD50 of the undigested group was 6.4 ng. Therefore, the LD50 of the enzyme-digested group of this invention is superior to that of naturally extracted botulinum toxin, and the LD50 of the enzyme-digested group is superior to that of the undigested group.

[0103] The present invention has been illustrated by the above embodiments; however, it should be understood that the present invention is not limited to the specific embodiments and implementations described herein. The purpose of including these specific embodiments and implementations herein is to assist those skilled in the art in practicing the present invention. Any person skilled in the art can easily make further improvements and modifications without departing from the spirit and scope of the present invention; therefore, the present invention is limited only by the content and scope of the claims, and is intended to cover all alternatives and equivalents included within the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A botulinum toxin A1 precursor molecule for preparing natural botulinum toxin A, characterized in that, The botulinum toxin A1 precursor molecule includes a light chain and a heavy chain, and a linker region connecting the light chain and the heavy chain. The amino acid sequence of the botulinum toxin A1 precursor molecule is shown in SEQ ID NO.

4. The light chain is the amino acid sequence from position 1 to position 444 of natural botulinum toxin A, as shown in SEQ ID NO.

5. The heavy chain sequence is the amino acid sequence from position 449 to position 1296 of natural botulinum toxin A, as shown in SEQ ID NO.

6. The amino acid sequence of the linker region is shown in SEQ ID NO.

11. The amino acid sequence of natural botulinum toxin A is shown in SEQ ID NO.

7.

2. The botulinum toxin A1 precursor molecule as described in claim 1, characterized in that, The botulinum toxin A1 precursor molecule has two protease Kex2 cleavage sites, which are located at KR positions 444 and 445, and KR positions 455 and 456, respectively, of the amino acid sequence shown in SEQ ID NO.

4.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the botulinum toxin A1 precursor molecule as described in claim 1 or 2, the nucleic acid molecule being shown in SEQ ID NO. 3, wherein the nucleic acid molecule encoding the light chain is shown in SEQ ID NO. 8, the nucleic acid molecule encoding the heavy chain is shown in SEQ ID NO. 9, and the nucleic acid molecule encoding the linker region is shown in SEQ ID NO.

10.

4. An engineered bacterium for producing the botulinum toxin A1 precursor molecule as described in claim 1 or 2, characterized in that, The engineered bacteria contain the nucleic acid molecules as described in claim 3.

5. The engineered bacteria of the botulinum toxin A1 precursor molecule as described in claim 4, characterized in that, The engineered bacteria is Escherichia coli.

6. A method for preparing the botulinum toxin A1 precursor molecule as described in claim 1 or 2, characterized in that, The preparation method includes the following steps: 1) The 000C(KK) amino acid sequence shown in SEQ ID NO.2 was inserted into a prokaryotic expression vector to obtain the 000C(KK) vector; 2) Using overlap extension PCR, all KK sites on the 000C(KK) amino acid sequence were mutated to KR, so that positions 445 to 456 of the 000C(KK) amino acid sequence were RHHHHHHGYNKR, and the KRs at both ends of the 6×His tag had two enzyme cleavage sites of the protease Kex2, so as to obtain the mutated sequence. as well as 3) The mutated sequence is ligated to the 000C(KK) vector to obtain a recombinant vector with the Top10-000C(2KR) sequence, the amino acid sequence of which is shown in SEQ ID NO.

4.

7. The preparation method according to claim 6, characterized in that, In step 1), the prokaryotic expression vector is pET28, pET32, pQE30, pQE60, or pGEX-6P.

8. A method for preparing natural botulinum toxin A using the botulinum toxin A1 precursor molecule as described in claim 1 or 2, the method comprising the following steps: 1) Transform the recombinant vector containing the amino acid sequence Top10-000C(2KR) shown in SEQ ID NO.4 into competent Escherichia coli cells to construct a genetically engineered bacterium expressing the target protein; 2) The target protein is expressed by fermentation culture of the genetically engineered bacteria, and after purification, botulinum toxin precursor protein is obtained; and 3) The botulinum toxin precursor protein was cleaved by Kex2 protease to remove the 6XHis tag and simultaneously activate it. Then, an extra arginine residue was removed from the C-terminus of the light chain by carboxypeptidase B to obtain mature natural botulinum toxin A as shown in SEQ ID NO.

7.

9. The method as described in claim 8, characterized in that, In step 1), the Escherichia coli is BL21, BL21(DE3), BL21(DE3)plysS, BLR, BLR(DE3), BLR(DE3)pLysS, Rosetta(DE3), Origami B(DE3), Origami2(DE3), Rosetta gami(DE3)pLysS, Rosetta gamiB(DE3), Rosetta gami 2(DE3), ShuffleT7-K12, Shuffle T7-B, or BL21 Star(DE3).

10. The method as described in claim 8, characterized in that, In step 2), the purification includes Ni column affinity chromatography, UniGel 30Q anion chromatography, Q anion chromatography, and SP cation chromatography.

11. The method as described in claim 8, characterized in that, In step 3), the Kex2 protease further includes a Kex2 digestion buffer, which is 50 mM Tris-HCl, pH 8.0, 0.1–2 mM CaCl2; or 50 mM Tris-HCl, pH 8.0, 0.1–2 mM CaCl2, 0.01%–0.5% Tween-20.

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