Polypeptide for efficiently and rapidly recombining and expressing neurotoxin protein and method thereof

Abstract

The polypeptide is used for preparing neurotoxin protein through recombinant expression and comprises a first restriction enzyme cutting site, a second restriction enzyme cutting site and a tag peptide fragment. The tag peptide fragment is used for affinity purification, the first restriction enzyme cutting site at least comprises one amino acid and is located on one side of the N end of the tag peptide fragment, and the second restriction enzyme cutting site at least comprises one amino acid and is located on one side of the C end of the tag peptide fragment. It is verified that after the polypeptide is used for fusion expression of neurotoxin, a large amount of neurotoxin target protein with biological activity can be rapidly prepared.

Description

Technical Field

[0001] The present invention relates to a polypeptide, in particular to a polypeptide containing an endonuclease cleavage site, which is used for fusion expression to prepare neurotoxins derived from organisms. Background Art

[0002] Neurotoxins are chemical substances that mainly act on ion channels. They can act on the junction of motor nerves and muscles, causing skeletal muscles to fail to contract. In severe cases, they can lead to respiratory paralysis and death from suffocation. Certain organisms in nature also contain or can release such toxins, such as botulinum neurotoxin (BoNT), tetanus neurotoxin (TeNT) and diphtheria toxin (Diphtheria Toxoid).

[0003] BoNT is known for its ability to cause muscle relaxation and paralysis. In particular, BoNT / A has been used in a variety of medical and cosmetic procedures, including the treatment of frown lines or hypermobility facial lines, migraines, hemifacial spasms, bladder hyperactivity, hyperhidrosis, nasolabial lines, cervical dystonia, blepharospasm and spasticity. Due to its strong neurotoxicity, the median lethal dose (LD50) is 50 ) is 1ng / kg, so the production and sales of botulinum toxin products in various countries are strictly controlled.

[0004] As of 2022, only two types of botulinum toxin, type A and type B, have been developed for commercial use. Type A is the most potent, with about 20 type A botulinum toxin products approved for marketing worldwide (Journal of Neural Transmission (2022) 129:829–833). Type B is mainly used for patients who have antibodies to type A botulinum toxin. Only one type B botulinum toxin product, MyoBloc produced by Solstice in the United States, has been approved for marketing by the US FDA (J Neurol 2003, 249:1729-1732).

[0005] At present, all the products on the market are derived from Clostridium botulinum toxin. On the one hand, due to the limitations of its toxicity, expression level and purification process, Clostridium botulinum toxin has a long production cycle, high cost and poor production process safety. On the other hand, there are multiple enzyme cleavage sites in the sequences of these neurotoxins, such as trypsin, enterokinase, glutamyl endopeptidase, chymosin and dibasic amino acid endopeptidase, so that in the process of fusion expression to produce these neurotoxins, they are easily cleaved by enzymes, resulting in relatively large polypeptide fragments, low yields, or it is difficult to obtain neurotoxic proteins.

[0006] In order to solve this problem, people have conducted many useful explorations and achieved some progress, such as: CN114957482 and CN114958887, but there is still a need to find a better solution. Summary of the invention

[0007] One object of the present invention is to provide a polypeptide for producing a neurotoxin derived from an organism by recombinant (eg fusion) expression, so as to obtain a protein with neurotoxicity.

[0008] Another object of the present invention is to provide a polypeptide for producing neurotoxins derived from organisms in recombinant (eg, fusion) expression, thereby increasing the yield of neurotoxins.

[0009] Another object of the present invention is to provide a polypeptide for producing neurotoxins derived from organisms by recombinant (eg fusion) expression, thereby shortening the production cycle and improving the production efficiency.

[0010] Another object of the present invention is to provide a method for preparing neurotoxin protein by recombinant expression, wherein the light chain and heavy chain of the neurotoxin are connected to the two ends of the artificial polypeptide sequence respectively, and recombinant expression is performed to improve the efficiency of the recombinant expression of the neurotoxin.

[0011] The neurotoxin referred to in the present invention is derived from microorganisms, and its target is mammals, including a heavy chain containing more amino acids and a light chain containing fewer amino acids, and a disulfide bond connecting the heavy chain and the light chain. The light chain contains an active domain that exerts the neurotoxin effect, and the heavy chain contains a receptor binding domain that binds to the receptor.

[0012] The neurotoxins of the present invention include, but are not limited to, botulinum neurotoxin and tetanus neurotoxin.

[0013] Botulinum neurotoxin should be understood to include seven serotypes, namely A, B, C, D, E, F and G. Among them, the four serotypes of botulinum neurotoxin A\B\C\D are from Clostridium botulinum, serotype E is from Clostridium butyricum, serotype F is from Clostridium baratii, and serotype G is from Clostridium argentinense. Studies have shown that the sequence variation between the subtypes of type A botulinum neurotoxin is very high, and there is only 76% homology between light chains A3 and A4. In the sequence of botulinum neurotoxin, there are also some conserved amino acids related to catalytic activity. For example: but not limited to His223, Glu224, His227, Glu261, Glu262, Glu351, Arg363, and Tyr366. (J. Mol. Biol. (2006) 362, 733-742) Therefore, according to the teachings, technicians use genetic engineering mutation methods (such as alanine scanning) to adjust other amino acids in the sequence to obtain artificial homologous sequences.

[0014] Tetanus neurotoxin comes from the bacterium Clostridium tetani.

[0015] The recombinant expression method for preparing neurotoxin protein of the present invention connects the light chain and heavy chain of neurotoxin to the two ends of artificial polypeptide sequence respectively to form a fusion expression sequence, and implements fusion expression in the expression system.

[0016] The protein expressed by the expression system is obtained by means of enzyme cutting, separation and purification to obtain a protein that is identical to the natural neurotoxin sequence or has a homology of more than 70%.

[0017] In some specific embodiments, the recombinantly expressed neurotoxin has greater than 75% homology to the natural neurotoxin.

[0018] In other specific embodiments, the recombinantly expressed neurotoxin has greater than 80% homology to the natural neurotoxin.

[0019] In other specific embodiments, the recombinantly expressed neurotoxin has a homology greater than 85% with the natural neurotoxin.

[0020] In other specific embodiments, the recombinantly expressed neurotoxin has greater than 90% homology to the natural neurotoxin.

[0021] In other specific embodiments, the recombinantly expressed neurotoxin has a homology greater than 95% with the natural neurotoxin.

[0022] In other specific embodiments, the recombinantly expressed neurotoxin has a homology greater than 97% with the natural neurotoxin.

[0023] In other specific embodiments, the recombinantly expressed neurotoxin has a homology greater than 99% with the natural neurotoxin.

[0024] Expression systems suitable for the method of the present invention include, but are not limited to, prokaryotic expression systems (such as, but not limited to, Escherichia coli and Bacillus subtilis), eukaryotic expression systems (such as yeast), insect rod-shaped expression systems (such as, but not limited to, SF9, SF21), or mammalian expression systems (such as, but not limited to, CHO cells and VERO cells). The present invention prefers prokaryotic expression systems, such as, for example, Escherichia coli, especially, strains such as BL21 (DE3), HMS174 (DE3), pLysS and pLysE.

[0025] A method for implementing the recombinant expression of the present invention using a plasmid as a vector is provided to facilitate the transfer of the recombinant sequence into an expression system.

[0026] Another specific embodiment of the expression plasmid is a plasmid used in the Bacillus subtilis expression system, such as but not limited to pHT series plasmids (such as but not limited to pHT01, pHT43, pHT304 and pHT44), pGAL1 series plasmids (such as but not limited to pGAL1 and pGAL10), pRS series plasmids (such as but not limited to pRS426), and p426 GAL, pYIP5, pZHY500, pYX212, pDNR1, pBLADE-SX, pYM39-EYFP, pYM30-ECFP, pFA6a, pYADE4, pP-αSUMO3, pGADT7AD, pRS425, pFA6a and YCplac22, etc.

[0027] Another specific embodiment of the expression plasmid is a plasmid used in a yeast expression system, such as but not limited to the pYES2 plasmid, the pPICZ series plasmids (such as but not limited to pPIC6αA, pPIC6αB, pPIC6αC, pPIC6 A, pPIC6 B), the pMA5 series plasmids, and pSTOP1622, pGFP315, pGFP22, pIL253, pAD43-25, pHCMC05 and pDG1363, etc.

[0028] Another specific embodiment of the expression plasmid is a plasmid used in an insect expression system, such as but not limited to pAcGP67 series plasmids, pFastBac series plasmids, and pIZT / V5-His, pFastBacHT A, pBacPAK8, pBacPAK8-His, pBacPAK9, pMT / BioEase-DEST, pVL1392, pVL1393, pXINSECT-DEST39, pIB / V5 and pAc5-V5-HisB, etc.

[0029] The polypeptide provided by the present invention is used to connect (e.g., covalently connect) a heavy chain and a light chain constituting a neurotoxin, with one end of the polypeptide connected to the light chain and the other end connected to the heavy chain. For example, the N segment of the polypeptide is covalently connected to the light chain, and the C segment is covalently connected to the heavy chain.

[0030] A specific embodiment of the polypeptide of the present invention includes:

[0031] Tag peptides, which are used for affinity purification;

[0032] The first restriction site, which includes at least one amino acid, is located at the N-terminal side of the tag peptide segment;

[0033] The second restriction enzyme cleavage site includes at least one amino acid and is located at the C-terminal side of the tag peptide segment.

[0034] Applicable tag peptides include, but are not limited to, FLAG, Myc, STREP, HA, or His-(Xm)n-His sequences, where m and n are selected from natural numbers, especially natural numbers from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. n indicates the number of amino acids Xm connected, for example, when n is 3, it indicates that Xm has 3 amino acids connected; when n is 5, it indicates that Xm has 5 amino acids connected. m indicates the position of each amino acid, for example, when n is 3, it indicates that Xm has 3 amino acids connected, (Xm)n is X1-X2-X3; when n is 3, it indicates that Xm has 3 amino acids connected, (Xm)n is X1-X2-X3-X4-X5. Each Xm is independently selected from natural amino acids, especially Lys, Met, Asp, Arg, Tyr, Glu, and His. For example, when n is 3, it means that Xm has 3 amino acids connected, and (Xm)n is X1-X2-X3, and X1, X2 and X3 are independently selected from natural amino acids, especially: Lys, Met, Asp, Arg, Tyr, Glu and His.

[0035] The tag peptide segment of the present invention preferably comprises a peptide formed by oligomerization of 6 to 12 histidines, such as HHHHHH, HHHHHHH or HHHHHHHHH.

[0036] The amino acid of the first restriction site is selected from arginine (R) and / or lysine (K), including but not limited to: R, K, RR, KK, RK or KR.

[0037] The amino acid of the second restriction site includes at least one K or R, such as: DDDDK, R, K, RR, KK, RK or KR.

[0038] Another specific embodiment of the polypeptide of the present invention includes:

[0039] Tag peptides, which are used for affinity purification;

[0040] The first restriction site, which includes at least one amino acid, is located at the N-terminal side of the tag peptide segment;

[0041] The second restriction site includes at least one amino acid and is located at the C-terminal side of the tag peptide segment;

[0042] The first restriction site is the dibasic endopeptidase (Kex2) restriction site.

[0043] Another specific embodiment of the polypeptide of the present invention includes:

[0044] Tag peptides, which are used for affinity purification;

[0045] The first restriction site, which includes at least one amino acid, is located at the N-terminal side of the tag peptide segment;

[0046] The second restriction site includes at least one amino acid and is located at the C-terminal side of the tag peptide segment;

[0047] The first restriction enzyme cutting site and the second restriction enzyme cutting site are Kex2 restriction enzyme cutting sites.

[0048] In order to increase the flexibility of the polypeptide of the present invention and to accelerate the speed of enzymatic cleavage by the protease, the N-terminus and / or C-segment of the tag peptide segment of the present application also includes at least one glycine (G) or serine (S), such as: G, S, GS, SG, GG, SS, GGS, SGG, GSG and SGS, etc.

[0049] Another specific embodiment of the polypeptide of the present invention includes:

[0050] Tag peptides, which are used for affinity purification;

[0051] The first restriction site, which includes at least one amino acid, is located at the N-terminal side of the tag peptide segment;

[0052] The second restriction site includes at least one amino acid and is located at the C-terminal side of the tag peptide segment;

[0053] The first flexible sequence is located between the tag peptide segment and the first restriction enzyme cleavage site or between the tag peptide segment and the second restriction enzyme cleavage site.

[0054] Another specific embodiment of the polypeptide of the present invention includes:

[0055] Tag peptides, which are used for affinity purification;

[0056] The first restriction site, which includes at least one amino acid, is located at the N-terminal side of the tag peptide segment;

[0057] The second restriction site includes at least one amino acid and is located at the C-terminal side of the tag peptide segment;

[0058] A first flexible sequence, which is located between the tag peptide segment and the first restriction site;

[0059] The second flexible sequence is located between the tag peptide segment and the second restriction enzyme cleavage site.

[0060] The polypeptide of the present invention also includes at least one auxiliary sequence for improving the cleavage efficiency of the endonuclease, such as but not limited to: GSGS, EEGSGS, GSGSDDGSGS, GSGSEDGSGS, GSGSDEGSGS, EEAE, DDAD, TEEAEKL, TDDADKL, TEDAEKL, TDEADKL, GTEEAEKLG, EGTEEAEKLG, EGTDDADKLG, EGTEEADKLG, EGTDDAEKLG, EGTEDADKLG and EGTDEAEKLG, as well as sequences with greater than 70% homology.

[0061] In some specific embodiments, the auxiliary sequences are greater than 75% homologous.

[0062] In other specific embodiments, the auxiliary sequences are more than 80% homologous.

[0063] In other specific embodiments, the auxiliary sequences are more than 85% homologous.

[0064] In other specific embodiments, the auxiliary sequences are more than 90% homologous.

[0065] In other specific embodiments, the auxiliary sequences are greater than 95% homologous.

[0066] Another specific embodiment of the polypeptide of the present invention includes:

[0067] Tag peptides, which are used for affinity purification;

[0068] The first restriction site, which includes at least one amino acid, is located at the N-terminal side of the tag peptide segment;

[0069] The second restriction site includes at least one amino acid and is located at the C-terminal side of the tag peptide segment;

[0070] The first auxiliary sequence is located between the tag peptide segment and the first restriction enzyme cleavage site or between the tag peptide segment and the second restriction enzyme cleavage site.

[0071] Another specific embodiment of the polypeptide of the present invention includes:

[0072] Tag peptides, which are used for affinity purification;

[0073] The first restriction site, which includes at least one amino acid, is located at the N-terminal side of the tag peptide segment;

[0074] The second restriction site includes at least one amino acid and is located at the C-terminal side of the tag peptide segment;

[0075] A first auxiliary sequence, which is located between the tag peptide segment and the first restriction enzyme cleavage site;

[0076] The second auxiliary sequence is located between the tag peptide segment and the second restriction enzyme cleavage site.

[0077] The polypeptide of the present invention is used for the fusion expression of neurotoxins, and the recombinant gene sequence for the fusion expression of neurotoxins is transferred into the expression system through a vector (such as a plasmid).

[0078] After the polypeptide of the present invention is used for the fusion expression of neurotoxins, the target neurotoxin protein with biological activity can be prepared quickly and in large quantities. The affinity tag used for purification is combined with the dibasic amino acid endopeptidase (Kex2) cleavage site, so that Kex2 selectively and preferentially acts on the polypeptide of the present invention to perform cleavage, avoiding Kex2 from performing cleavage on the neurotoxin protein sequence, not only improving the cleavage efficiency of the fusion expression protein, but also improving the integrity of the fusion expression neurotoxin protein, and improving the protein yield and activity.

[0079] Carboxypeptidase is used to enzymatically cut the K or R remaining at the C-terminus after Kex2 enzymatic cleavage, so that the neurotoxin produced by the prepared fusion expression no longer contains exogenous amino acids or polypeptides, thereby improving safety.

[0080] The polypeptide constructed by the present invention replaces the natural sequence between the light chain and the heavy chain. On the one hand, the expressed recombinant neurotoxin protein is in a non-toxic state before the Kex2 enzyme cleavage step, which greatly improves the safety of the production process. On the other hand, it also avoids the disordered cleavage of the natural sequence by cellular proteases, greatly reduces the polymorphism of the protein, improves the uniformity of the produced neurotoxin protein, and facilitates the implementation of subsequent protein purification, and the yield, purity and toxicity of the target protein are greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 Schematic diagram of the structure of recombinant botulinum toxin A protein and its expression vector; wherein A is a schematic diagram of the structure of recombinant botulinum toxin A protein, B is a predicted map of the surface accessibility of the Kex2 enzyme and the restriction sites in the recombinant botulinum toxin A protein, and C is a schematic diagram of the expression vector plasmid into which the recombinant botulinum toxin gene sequence is inserted;

[0082] Figure 2 The figure shows the results of recombinant botulinum toxin purification by nickel affinity chromatography and enzyme cleavage stability analysis; A is the electrophoresis diagram of the nickel affinity chromatography purification result, the arrow indicates the recombinant botulinum toxin, B is the electrophoresis diagram of the Kex2 protease enzyme cleavage purification sample; C is the Western blot diagram of the enzyme cleavage result, the sample before enzyme cleavage (0min) containing the HIS tag is loaded with 100ng, and the sample loading amount of other samples is 3μg. In each figure, M represents the protein molecular weight marker;

[0083] Figure 3 The results of recombinant botulinum toxin after enzyme digestion and second nickel column affinity chromatography purification; A is the reduced SDS-PAGE electrophoresis analysis after enzyme digestion, B is the Western blot method to detect the effect of enzyme digestion (the sample loading amount of the protein with HIS tag before enzyme digestion is 30ng, and the loading amount of other samples is 3μg), C is the reduced SDS-PAGE electrophoresis of nickel column affinity chromatography after enzyme digestion; D is the electrophoresis of recombinant protein with natural botulinum toxin 438-448 sequence (treated at room temperature for 3h and run SDS-PAGE electrophoresis), in each figure, M is the protein molecular weight marker;

[0084] Figure 4 Figures 1 and 2 are the results of purification by anion HQ column chromatography; A is the electrophoresis diagram of the separation and purification results by HQ column; B is the electrophoresis diagram of the separation and purification results by Superdex 200 column. In each figure, M is the protein molecular weight marker.

[0085] Figure 5 The purity and HIS residue detection results of the prepared recombinant botulinum toxin are shown in Figure 1; A is the SDA-PAGE electrophoresis diagram, B is the Western blot analysis diagram (the "positive" sample is the purified, non-enzyme-cleaved, HIS-tagged recombinant botulinum toxin protein), and C is the HPLC chromatogram. In each figure, M is the protein molecular weight marker;

[0086] Figure 6 The primary structure detection result diagram of the recombinant botulinum toxin prepared by mass spectrometry analysis; wherein A is the detection result diagram of the protein's precise molecular weight, B is the detection result diagram of the C430 / 454 disulfide bond position, C is the detection result diagram of the C1235 / 1280 disulfide bond position, and D is the detection result diagram of the protein's N- and C-terminal amino acid sequences;

[0087] Figure 7 These are the toxicity test results of the recombinant botulinum toxin prepared in this example; wherein A is the toxicity result diagram of the botulinum toxin recombinant protein A injected through the tail vein; B is the toxicity result diagram of the botulinum toxin recombinant protein A injected through the peritoneal method; and C is the toxicity test result of the recombinant botulinum toxin A protein with various auxiliary sequences after activation. DETAILED DESCRIPTION

[0088] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0089] Unless otherwise specified, all other reagents used in the embodiments of the present invention were purchased from China National Pharmaceutical Group Corporation or MERCK Corporation of the United States.

[0090] Example 1 Construction of recombinant botulinum toxin A protein and engineered bacteria

[0091] like Figure 1 A shows the schematic diagram of the structure of the recombinant botulinum toxin A protein, which includes the first restriction site, the first auxiliary sequence, the tag peptide, the second auxiliary sequence and the second restriction site between the light chain and the heavy chain. The polypeptide used in this example to connect the light chain (SEQ ID No 1) and the heavy chain (SEQ ID No 2) of botulinum toxin A has its N segment connected to the light chain and its C segment connected to the heavy chain. The sequence from its N-terminus to the C-terminus is constructed as follows:

[0092] Kex2 protease cleavage site (KR) - first auxiliary sequence (SEQ ID No 3) - tag peptide (composed of 8 histidine HIS) - second auxiliary sequence (SEQ ID No 3) - Kex2 protease cleavage site (KR).

[0093] The fusion protein (SEQ ID No 4) has 9 Kex2 protease cleavage sites, 7 of which belong to the natural protein sequence, but the accessibility of these sites to proteases is much lower than that of the protease cleavage sites in the linker peptide, such as Figure 1 As shown in B.

[0094] The coding gene sequence of the recombinant botulinum toxin protein sequence was designed and optimized according to the characteristics of the host Escherichia coli as shown in SEQ ID No 5, and the gene sequence was inserted into the pET-28 plasmid to obtain the expression vector pET-28-RDS, as shown in Figure 1C. After sequencing verification, the sequence was transformed into the host bacterium E. coli BL-21 (DE3) (direct source and original source were both from ThermoFisher Scientific, USA).

[0095] Example 2 Preparation of active recombinant botulinum toxin protein

[0096] The engineered bacteria were cultured in a shake flask, and the expression of recombinant botulinum toxin was induced by 0.3mM IPTG. The fermentation broth was centrifuged to obtain the bacterial cells, and then the cells were broken by ultrasonic wave. The recombinant botulinum toxin protein was initially purified by nickel column affinity chromatography. The non-natural botulinum toxin amino acid sequence in the obtained protein was removed by two proteases, Kex2 and carboxypeptidase B, and then a variety of separation methods such as ion exchange chromatography and molecular sieve chromatography were used to obtain a high-purity target protein containing only the natural botulinum toxin amino acid sequence, with a purity of more than 99%. The specific separation and purification process is as follows:

[0097] (1) Bacteria fragmentation

[0098] The cells obtained by centrifugation were resuspended by adding 3 times the volume of NaCl (20g / L), and the cells were collected after centrifugation at 4°C, 8000rpm for 10min. After obtaining the cells, 9 times the volume of buffer (50mM phosphate, 0.1MNaCl, pH=7.0) was added to resuspend the cells, and 5‰ PMSF (100mM) was added. Under low temperature conditions, a high-pressure homogenizer (Shanghai Hongli Technology Biological Co., Ltd., 850bar, 2 times) was used to break the cells, and the lysate was centrifuged at 12,000rpm for 40min, the supernatant was collected, and the sample solution was filtered with a 0.45μm filter head to obtain the sample solution.

[0099] (2) Nickel affinity chromatography

[0100] In a chromatography cabinet at 4°C, the sample solution was passed through a pre-equilibrated nickel affinity chromatography column (Biyuntian Biotechnology Co., Ltd.), and then washed with 5 column volumes of equilibrium buffer (50mM phosphate, 0.1M NaCl, pH=7.0) until no protein solution flowed out, and then eluted with a step-by-step gradient. The eluents were solutions containing 20, 50, 200, and 500mM imidazole prepared with the equilibrium buffer (each gradient was 5 column volumes). The elution peaks of each section were collected separately, and the protein purity and content of each peak were detected by SDS-PAGE electrophoresis. The experimental results showed that the recombinant botulinum toxin protein was mainly present in the elution solution of 50mM imidazole, with a high purity, see Figure 2A. The eluate of 50mM imidazole was collected, ultrafiltered 100 times with 50mM phosphate buffer (pH=7.0), Kex2 was added at a ratio of 1:500 (enzyme: protein), incubated for 5 hours, and samples were taken regularly for electrophoresis to detect the non-specific enzymatic cleavage effect of the protease on the target protein. The results showed that after 5 hours of enzymatic cleavage, the amount of the target protein did not change much, indicating that the non-specific enzymatic cleavage effect of Kex2 on the target protein was very weak, see Figure 2 B. At the same time, the Western blot method was used to detect the effect of HIS tag removal. The results showed that as the incubation time increased, the HIS tag of the target protein gradually decreased. After 3 hours of enzyme digestion (sample loading 3μg), the reduction rate was significantly reduced. At this time, the band intensity was weaker than that of the sample before enzyme digestion (sample loading 100ng), which indicated that after 3 hours of incubation with the protease, more than 96% of the target protein was removed from the HIS tag. Figure 2 C.

[0101] It can be seen that this example has verified that the scheme of using Kex2 protease to remove exogenous polypeptides is feasible.

[0102] (3) Removal of the connecting peptide

[0103] The eluate containing a large amount of target protein after nickel column chromatography was collected and concentrated and ultrafiltered using a 50K ultrafiltration centrifuge tube (Sartorius). The specific operation was to concentrate the protein eluate to 1 / 6 of the original volume, and then use an enzyme cleavage buffer (50mM phosphate, 0.1M NaCl, pH=7.0) to dilute the protein concentrate 6 times, repeat the concentration and dilution once, and then concentrate the solution to a protein concentration of about 2mg / ml. KEX2 protease and carboxypeptidase B (Shanghai Yaxin Biotechnology Co., Ltd.) were added at a mass ratio of 1:500 (enzyme: protein). Enzyme cleavage buffer was added to make the botulinum toxin protein concentration at 0.5mg / ml, and enzyme cleavage was performed at 25°C for 3 hours. The sample after enzyme cleavage (nickel column loading solution) was subjected to reduction electrophoresis. The results showed that the protein after enzyme cleavage was mainly 100KDa and 50KDa, while the sample protein molecular weight without protease was 150KDa, indicating that the protease cleaved the target protein. At the same time, it was shown that the protein before enzyme cleavage was a single chain and non-toxic, see Figure 3 A. Western blot was used to further detect the removal of the HIS tag. The results showed that although the loading amount of the sample after 3 hours of digestion (3 μg) was 100 times that of the positive protein (30 ng), the intensity of the target band of the former was lower than that of the latter, indicating that more than 99% of the HIS tag was removed. Figure 3 B.

[0104] After the digestion was completed, the digestion solution was passed through a pre-equilibrated nickel affinity chromatography column (column equilibration solution was 50 mM phosphate buffer, pH = 7.0) once at low temperature, the flow-through and washing solution (3 times the column volume) were collected, EDTA (final concentration was 1 mM) was added, and the digestion effect was detected by Western blot and SDS-PAGE. The SDS-PAGE results showed that the protein after digestion was mainly in the effluent, and there were few target protein bands in the eluate, indicating that most of the proteins did not carry a HIS tag. The Western blot test results also proved this point, see Figure 3 B and 3C.

[0105] The recombinant protein of this example does not carry the natural botulinum toxin 439-448 sequence. This sequence will be removed after expression and secretion in Bacillus subtilis, and when prepared in the Escherichia coli expression system, it will be non-specifically removed, and the removal site is unclear. Our experimental results also confirmed this point, that is, the recombinant botulinum toxin protein with this sequence was prepared by the same method and digested by enzymes. The results showed that without adding protease, the recombinant botulinum toxin A protein can also be broken into two chains, see Figure 3 D.

[0106] (4) Anion exchange column chromatography

[0107] Under low temperature, the flow-through and washing liquid collected after the enzyme cleavage is completed and passed through the nickel affinity chromatography column are passed through the pre-equilibrated anion exchange column HQ (the column equilibration liquid is 50mM phosphate buffer, pH=7.0). After loading, it is rinsed with the equilibration buffer for 5-10 column volumes, and the flow-through peak is collected; then it is eluted with a linear gradient of 0-1mol / L NaCl, and the total elution volume is 20 column volumes. The elution peak appears after about 5 column volumes. The flow-through, washing liquid and eluent are collected, and the purity and content of the recombinant botulinum toxin protein in each sample are detected by SDS-PAGE electrophoresis. The experimental results show that the eluent is mainly impurities, and the botulinum toxin protein mainly appears in the flow-through with a purity of about 95%. Figure 4 A. The collected flow-through was concentrated to a protein concentration of 5 mg / ml using a 50K ultrafiltration centrifuge tube (Sartorius), and 10% glycerol was added and stored at -70°C until use.

[0108] (5) Superdex 200 gel filtration column chromatography

[0109] Under low temperature, the concentrated sample was passed through a pre-equilibrated (50mM phosphate, 0.1M NaCl, pH=7.0) Superdex 200 column (Cytiva), and eluted with equilibrated buffer at a flow rate of 0.5ml / min for 2 column volumes until no peak appeared. The elution peak appeared about 23min later. Each elution peak was collected separately, and the protein purity and content of each peak were detected by SDS-PAGE electrophoresis. The experimental results showed that the botulinum toxin protein was mainly concentrated in elution peak I, and there was basically no impurity band. The elution peaks II and III were mainly 55KDa and 30KDa impurity bands, respectively. Figure 4 B.

[0110] (6) Sample quantification and preservation

[0111] Collect the molecular sieve chromatography elution peak I, add 10% glycerol, and quantify it using the A280 and BCA methods to set the final protein concentration at 1-2 mg / mL. The purity and HIS tag residue of the final product were detected by SDS-PAGE, HPLC and WB. The results showed that the electrophoresis of the samples showed a single band, that is, they were electrophoretically pure. The WB results showed that the HIS tag residue was less than 1‰, and the HPLC test purity was 99.19%. Figure 5 The samples were stored in a -70℃ refrigerator.

[0112] Example 3 Sequence Identification of Recombinant Botulinum Toxin A Protein

[0113] The botulinum toxin protein is composed of two polypeptide chains, which are connected by a pair of interchain disulfide bonds. The protein sample was centrifuged at 12000g for 30 minutes without adding or adding the reducing agent TCEP (TCEP can open the interchain disulfide bonds, separate the two chains, and put the sample in a reduced state) to the sample, and then separated by a chromatographic column ACQUITY UPLC Protein BEH 300C4 column and entered into a Xevo G2-XS Q-TOF (Waters) mass spectrometer to determine the intact molecular weight and reduced molecular weight of the botulinum toxin protein prepared in this example. The mass spectrometry data was analyzed after deconvolution by UNIFI software, and the specific data is shown in Figure A. The results show that the measured molecular weights of the full-length, light chain and heavy chain of the botulinum toxin protein prepared in this example are 148049.4Da (theoretical value 148045.3Da), 49897.5Da (theoretical value 49896.1Da) and 98151.9Da (theoretical value 98151.2Da), respectively, and the deviations from the theoretical molecular weight are all less than 28ppm, indicating that the molecular weight of the botulinum toxin protein prepared in this example is consistent with the theoretical value. This indicates that the amino acid sequence of the obtained recombinant botulinum toxin protein is the same as the designed one.

[0114] Disulfide bonds are an important form of post-translational modification of proteins. Inter-chain or intra-chain disulfide bonds are essential for protein molecules to maintain the correct higher-order structure and necessary biological activity. Nanoliter liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) was used to identify the intra-chain and inter-chain disulfide bond pairing patterns of the botulinum toxin protein prepared in this embodiment. The nanoliter liquid chromatography and mass spectrometer are EASY-nLC 1200 and Thermo OrbitrapQExactive HF from Thermo Corporation of the United States, respectively, and the liquid analysis column is a C18 reverse phase column. The mass spectrometry data was analyzed by BioPharma Finder (V5.2) software, and rich primary and secondary ion information was collected for each pair of disulfide bonds. The primary mass deviation of the ion signal value was 10ppm, and the secondary mass deviation was 20ppm. The results show ( Figure 7 -B), the 429th cysteine ​​of the light chain forms a disulfide bond with the 6th cysteine ​​of the heavy chain (i.e. the disulfide bond formed by the 430th and 454th cysteine ​​of the natural botulinum toxin protein), and the 787th cysteine ​​of the heavy chain also forms a disulfide bond with the 832nd cysteine ​​(i.e. the disulfide bond formed by the 1235th and 1280th cysteine ​​of the natural botulinum toxin protein). For detailed values, see Figure 7 -C. The results show that the botulinum toxin protein prepared in this example contains a pair of interchain disulfide bonds and a pair of intrachain disulfide bonds, and the disulfide bond connection mode is the same as the theory.

[0115] The amino acid sequence is the basis for protein function. Figure 6 -A The results indicate that the amino acid sequence of the recombinant botulinum toxin protein we obtained is the same as the designed one. In order to further verify this result, the C- and N-terminal sequences of the protein were detected. The protein sample prepared in this example was denatured with guanidine hydrochloride, reduced, and hydrolyzed with protease Glu-C and Trypsin, and then separated by a chromatographic column ACQUITY UPLC Protein BEH 300C4 column and entered into the Xevo G2-XS Q-TOF (Waters) mass spectrometer to determine the N- and C-terminal amino acid sequences of the protein, and the mass spectrometry data were analyzed using UNIFI software. The results showed that the N- and C-terminal amino acid sequences of the light chain of the botulinum toxin protein prepared in this example were PFVNK and LLCVRGIITS, respectively, and the N- and C-terminal amino acid sequences of the heavy chain were ALDLCIK and VDDGWGERPL, respectively, which were consistent with the theoretical sequence, see Figure 6 D. This also indicates that after enzyme digestion, all foreign amino acid sequences are removed.

[0116] Example 4 Toxicity determination of recombinant botulinum toxin A protein

[0117] Take 5 26-30 day old SPF grade Kunming mice, inject 0.1 ml of the prepared recombinant botulinum toxin product into each mouse through the tail vein, the concentration is 10ug / ml, calculate the average death time (in minutes), and calculate the sample toxicity according to the formula Y = 17776578-868930×t+14382×t2-78.90×t3, t is the death time in minutes, Y is the corresponding toxicity calculated by the regression equation, and a total of three batches were tested. The results showed that the LD50 (pg / 25g, U) of the prepared recombinant botulinum toxin measured twice were 5.48 and 5.64, respectively, that is, the average toxicity unit of the recombinant botulinum toxin was 5.56pg / U, and the average toxicity unit (U) of each mg of the product to Kunming mice was 18×10 7 ,See Figure 7 A.

[0118] Intraperitoneal injection:

[0119] The sample was diluted in an arithmetic series and injected intraperitoneally into 5 female Kunming mice aged 26 to 30 days using a disposable sterile syringe for each dilution, 0.5 ml / mouse. The animals were observed for disease and death at least once a day after injection for 4 consecutive days, and the death of mice was recorded. A total of three batches were tested. According to the number of deaths of experimental animals within 4 days, the toxicity LD50 of the sample was calculated using the statistical method Reed Muench method, and the calculation formula was:

[0120]

[0121] The results showed that the LD50 (pg / 25g, U) of the two measurements were 5.3 and 6.2, respectively, that is, the average toxicity unit of the recombinant botulinum toxin was 5.75 pg / U, and the number of toxicity units (U) per mg of the product for Kunming mice was 17.4×10 7 ,See Figure 7 B. The results obtained by the two toxicity detection methods are similar. The toxicity of the botulinum toxin protein prepared in this example is much higher than the minimum value of 1×10 7 U / mg, further indicating that the primary and higher structures of botulinum toxin protein, such as amino acid sequence, disulfide bond position, and spatial folding, are identical to those of the natural ones.

[0122] The above data show that the connecting peptide designed in this patent can ensure that the botulinum toxin forms the correct disulfide bond, so that the recombinant botulinum toxin protein has a high biological activity. On the basis of the above examples, several other auxiliary sequences (the polypeptide sequence between the first restriction site and the second restriction site, also referred to as the interenzyme polypeptide in this example) are also used, and the corresponding expression clones are constructed and the target protein is prepared and activated, and then the effects of these auxiliary sequences and tag peptides on the biological activity of the recombinant botulinum toxin are analyzed (SEQ ID No 6 to SEQ ID No 12). The results show that these flexible polypeptides can maintain the toxicity of the recombinant botulinum toxin at a very high level (12.7×10 7 ~17.6×10 7 U / mg), indicating that the use of these interenzyme peptides can obtain recombinant botulinum toxin proteins with high biological activity. Figure 7 C.

Claims

1. A polypeptide for recombinant expression to produce a neurotoxin protein, characterized in that Tag peptides, which are used for affinity purification; The first restriction site, which includes at least one amino acid, is located at the N-terminal side of the tag peptide segment; The second restriction enzyme cleavage site includes at least one amino acid and is located at the C-terminal side of the tag peptide segment.

2. The polypeptide according to claim 1, characterized in that The tag peptide segment has a His-(Xm)n-His sequence, and m and n are selected from natural numbers respectively.

3. The polypeptide according to claim 2, characterized in that m and n are selected from natural numbers ranging from 1 to 10 respectively.

4. The polypeptide according to claim 2, characterized in that Each of the Xm is independently selected from Lys, Met, Asp, Arg, Tyr, Glu and His.

5. The polypeptide according to claim 2, characterized in that The tag peptide is selected from HHHHHH, HHHHHHH or HHHHHHHHH.

6. The polypeptide according to claim 1, characterized in that The amino acid of the first cleavage site is selected from at least one of arginine and lysine.

7. The polypeptide according to claim 1, characterized in that The first restriction enzyme cleavage site is selected from R, K, RR, KK, RK or KR.

8. The polypeptide according to claim 1, characterized in that The amino acids of the second restriction site include at least one arginine or lysine.

9. The polypeptide according to claim 1, characterized in that The second restriction enzyme cleavage site is selected from R, K, RR, KK, RK, KR or DDDDK.

10. The polypeptide according to claim 1, characterized in that The first restriction enzyme cleavage site is a Kex2 restriction enzyme cleavage site.

11. The polypeptide according to claim 1, characterized in that The first restriction enzyme cleavage site and the second restriction enzyme cleavage site are Kex2 restriction enzyme cleavage sites.

12. The polypeptide according to claim 1, characterized in that The tag peptide segment also includes a flexible sequence located at one or more of the N-terminus and the C-terminus of the tag peptide segment.

13. The polypeptide according to claim 1, characterized in that The flexible sequence includes at least one glycine or serine.

14. The polypeptide according to claim 13, characterized in that The flexible sequence is selected from G, S, GS, SG, GG, SS, GGS, SGG, GSG and SGS.

15. The polypeptide according to claim 13, characterized in that The flexible sequence is located between the tag peptide segment and the first enzyme cleavage site.

16. The polypeptide according to claim 13, characterized in that The flexible sequence is located between the tag peptide segment and the second enzyme cleavage site.

17. The polypeptide according to claim 1, characterized in that It also includes that the first auxiliary sequence is located between the tag peptide segment and the first enzyme cleavage site or between the tag peptide segment and the second enzyme cleavage site.

18. The polypeptide according to claim 17, characterized in that The auxiliary sequence is selected from GSGS, EEGSGS, GSGSDDGSGS, GSGSEDGSGS, GSGSDEGSGS, EEAE, DDAD, TEEAEKL, TDDADKL, TEDAEKL, TDEADKL, GTEEAEKLG, EGTEEAEKLG, EGTDDADKLG, EGTEEADKLG, EGTDDAEKLG or EGTEDADKLG and EGTDEAEKLG and homologous sequences with a homology greater than 70%.

19. The polypeptide according to claim 1, characterized in that Also includes: A first auxiliary sequence, which is located between the tag peptide segment and the first restriction enzyme cleavage site; as well as The second auxiliary sequence is located between the tag peptide segment and the second restriction enzyme cleavage site.

20. The polypeptide according to claim 19, characterized in that The first auxiliary sequence and the second auxiliary sequence are independently selected from GSGS, EEGSGS, GSGSDDGSGS, GSGSEDGSGS, GSGSDEGSGS, EEAE, DDAD, TEEAEKL, TDDADKL, TEDAEKL, TDEADKL, GTEEAEKLG, EGTTEEAEKLG, EGTDDADKLG, EGTEEADKLG, EGTDDAEKLG or EGTEDADKLG and EGTDEAEKLG and homologous sequences with a homology greater than 70%.

21. The polypeptide according to claim 1, characterized in that The polypeptide is used to connect a heavy chain and a light chain constituting a neurotoxin, the light chain is connected to one end of the polypeptide, and the heavy chain is connected to the other end of the polypeptide.

22. The polypeptide according to claim 1, characterized in that The polypeptide is used to connect a heavy chain and a light chain constituting a neurotoxin, the light chain is connected to the N-terminus of the polypeptide, and the heavy chain is connected to the C-terminus of the polypeptide.

23. The polypeptide according to claim 1, characterized in that Used for fusion expression of neurotoxins, the light chain of the neurotoxin is connected to the N-terminus of the polypeptide, and the heavy chain is connected to the C-terminus of the polypeptide.

24. The polypeptide according to claim 1, characterized in that The fusion expression is carried out in various expression systems, and the expression system is selected from a prokaryotic expression system, a eukaryotic expression system, an insect rod expression system or a mammalian expression system.

25. The polypeptide according to claim 1, characterized in that The prokaryotic organism is Escherichia coli.

26. The polypeptide according to claim 25, characterized in that The Escherichia coli is selected from BL21 (DE3), HMS174 (DE3), pLysS or pLysE.

27. The polypeptide according to claim 1, characterized in that The polypeptide is used to connect a heavy chain and a light chain constituting a botulinum neurotoxin, wherein the light chain of the botulinum neurotoxin is connected to the N-terminus of the polypeptide, and the heavy chain is connected to the C-terminus of the polypeptide; or The polypeptide is used to connect a heavy chain and a light chain constituting tetanus neurotoxin, wherein the light chain of tetanus neurotoxin is connected to the N-terminus of the polypeptide, and the heavy chain is connected to the C-terminus of the polypeptide.

28. The polypeptide according to claim 27, characterized in that The tag peptide segment is selected from HHHHHH, HHHHHHH or HHHHHHHHH; the first restriction enzyme cleavage site is a Kex2 restriction enzyme cleavage site; the second restriction enzyme cleavage site is a Kex2 restriction enzyme cleavage site or an enterokinase restriction enzyme cleavage site.

29. The polypeptide according to claim 28, characterized in that The amino acid sequence of the first restriction site is selected from R, K, RR, KK, RK or KR.

30. The polypeptide according to claim 28, characterized in that The amino acid sequence of the second restriction site is selected from DDDDK, R, K, RR, KK, RK or KR.

31. The polypeptide according to claim 27, characterized in that It also includes a flexible sequence located at one or several of the N-terminus and the C-terminus of the tag peptide segment, and is selected from G, S, GS, SG, GG, SS, GGS, SGG, GSG or SGS.

32. The polypeptide according to claim 27, characterized in that It also includes an auxiliary sequence, which is located between the tag peptide segment and the first restriction enzyme cleavage site, and at least one between the tag peptide segment and the second restriction enzyme cleavage site.

33. The polypeptide according to claim 27, characterized in that The auxiliary sequence is selected from GSGS, EEGSGS, GSGSDDGSGS, GSGSEDGSGS, GSGSDEGSGS, EEAE, DDAD, TEEAEKL, TDDADKL, TEDAEKL, TDEADKL, GTEEAEKLG, EGTEEAEKLG, EGTDDADKLG, EGTEEADKLG, EGTDDAEKLG or EGTEDADKLG and EGTDEAEKLG and homologous sequences with a homology greater than 70%.

34. A method for producing neurotoxin protein by recombinant expression, characterized in that The light chain and heavy chain of the neurotoxin are connected to the two ends of the artificial polypeptide sequence to form a fusion expression sequence, and the fusion expression is carried out in the expression system. The protein expressed by the expression system is subjected to enzyme digestion and separation and purification to obtain a protein that is identical to the natural neurotoxin sequence or has a homology of more than 70%; The artificial polypeptide comprises the polypeptide according to claim 1.

35. The method for producing neurotoxin protein by recombinant expression according to claim 34, characterized in that The expression system is selected from a prokaryotic expression system, a eukaryotic expression system, an insect rod-shaped expression system or a mammalian expression system.

36. The method for producing neurotoxin protein by recombinant expression according to claim 34, characterized in that The expression system is Escherichia coli.

37. The method for producing neurotoxin protein by recombinant expression according to claim 34, characterized in that The first restriction site of the polypeptide is a dibasic amino acid endopeptidase restriction site, and the tag peptide segment is selected from FLAG, Myc, STREP, HA or has a His-(Xm)n-His sequence, wherein m and n are respectively selected from natural numbers.

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