Recombinant TEV protease, recombinant vector and expression and purification method

Abstract

The invention provides a recombinant TEV protease, a recombinant vector and an expression and purification method, and relates to the technical field of gene engineering. According to the recombinant TEV protease, seven specific mutation sites are designed on the basis of original TEV protease, and the mutation sites reduce the intramolecular self-hydrolysis rate of the recombinant TEV protease and enhance the stability of a protein structure; meanwhile, the effective life of the protein is prolonged, and the stability of the protein under various application conditions is improved. The invention further provides a recombinant vector, the recombinant vector comprises an initial vector and the coding gene of the recombinant TEV protease, the initial vector contains the SUMO3 tag, the solubility of target protein can be enhanced, and the risk of forming insoluble aggregates in the expression process is reduced. The prokaryotic system is used for expressing the recombinant TEV protease, so that the TEV protease can be produced on a large scale, and the overall yield of the protein is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant TEV protease, a recombinant vector and an expression and purification method. Background Art

[0002] At present, recombinant tobacco etch virus (TEV) protease is widely used in protein engineering and biopharmaceuticals due to its high specificity and wide application. Because TEV protease can specifically recognize the heptapeptide sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly / Ser, and its sequence specificity is much higher than that of proteases such as Thrombin, FactorXa, and Enterokinase, and it can tolerate a variety of common protease inhibitors, and its enzymatic cleavage efficiency is high even at 4°C, it is particularly suitable for removing fusion tags. However, there are still some obvious defects in the existing TEV protease expression, purification, and activity. First, natural TEV protease is prone to intramolecular self-hydrolysis, resulting in truncation of the protease itself and a significant reduction in activity, affecting the application stability. Although studies have attempted to improve its stability through single-point mutations (such as S219N, S219V), these improvements have limited effect in improving solubility and yield. A large amount of protein is still precipitated in the form of insoluble inclusion bodies, and there is still a bottleneck in the comprehensive improvement of high activity and high stability. Summary of the invention

[0003] In view of this, an object of the present invention is to provide a recombinant TEV protease, which shows significant advantages in expression yield, stability and activity.

[0004] Another object of the present invention is to provide a recombinant vector containing a SUMO tag, which is fused with TEVMP7, which not only greatly improves the solubility of the protein, reduces the formation of insoluble aggregates, but also simplifies the purification process.

[0005] Another object of the present invention is to provide a method for expressing and purifying recombinant TEV protease, which can improve the purity and yield of recombinant TEV protease.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a recombinant TEV protease, the amino acid sequence of the recombinant TEV protease is shown as SEQ ID NO: 1.

[0008] Preferably, compared with the wild-type TEV protease, the recombinant TEV protease has an amino acid residue at position 17 mutated from threonine to serine, an amino acid residue at position 56 mutated from leucine to valine, an amino acid residue at position 68 mutated from asparagine to aspartic acid, an amino acid residue at position 77 mutated from isoleucine to valine, an amino acid residue at position 106 mutated from glutamic acid to glycine, an amino acid residue at position 135 mutated from serine to glycine, and an amino acid residue at position 219 mutated from serine to valine.

[0009] The present invention also provides a gene encoding the recombinant TEV protease, and the nucleotide sequence of the gene encoding the recombinant TEV protease is shown in SEQ ID NO:2.

[0010] The present invention also provides a recombinant vector for expressing TEV protease, wherein the recombinant vector comprises an initial vector and a gene encoding the recombinant TEV protease.

[0011] Preferably, the initial vector is PET21M-SUMO3.

[0012] The present invention also provides a method for expressing and purifying a recombinant TEV protease, comprising the following steps: transfecting the recombinant vector into Escherichia coli, selecting positive clones in the transfected Escherichia coli and inducing expression in a culture medium; collecting bacterial cells by centrifugation, and lysing the collected bacterial cells using a resuspension buffer to obtain a lysate; centrifuging the lysate to collect the supernatant; and purifying the supernatant to obtain the recombinant TEV protease.

[0013] Preferably, the Escherichia coli is BL21-CodonPlus(DE3)-RIPL.

[0014] Preferably, the culture medium is selected from LB medium containing 100 μg / mL ampicillin.

[0015] Preferably, the induction expression condition is 22-25° C. for 15-18 hours.

[0016] Preferably, the supernatant is purified using a Ni-NTA affinity column.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a recombinant TEV protease, which is designed and optimized with seven mutation sites based on the original TEV protease, and is named TEVMP7. Compared with the wild-type TEV protease (TEV) and the single mutant (TTEV), the recombinant TEV protease TEVMP7 of the present invention shows significant advantages in expression yield, stability and activity. TEVMP7 combines the optimized design of seven mutation sites, significantly slows down the autohydrolysis rate, greatly improves the stability of the protein structure, and more importantly, it can maintain high activity under a wide range of application conditions, significantly improving its applicability in various biochemical and industrial processes.

[0019] The present invention also provides a recombinant vector containing a SUMO tag, which is fused with TEVMP7, which not only greatly improves the solubility of the protein, reduces the formation of insoluble aggregates, simplifies the purification process, but also ensures that TEVMP7 remains highly active even when the SUMO tag is not removed. This design improves the versatility of TEVMP7, making it more adaptable and flexible in protein research and enzyme applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the plasmid map of PET21M-SUMO3;

[0021] Figure 2 This is the PET21M-SUMO3-TEVMP7 recombinant plasmid map of the present invention;

[0022] Figure 3 This is the SDS-PAGE run of TEVMP7;

[0023] Figure 4 is the collected peak chromatogram of TEVMP7;

[0024] Figure 5 The Image J software of TEVMP7 selected a part of the SDS-PAGE image for software calculation and illustration;

[0025] Figure 6 This is the SDS-PAGE gel image of TEV;

[0026] Figure 7 is the collected peak chromatogram of TEV;

[0027] Figure 8 Select a part of the SDS-PAGE image for TEV's Image J software to calculate the image;

[0028] Fig. 9The results of the comparative experiment of the enzymatic activities of the wild-type protease TEV, the single mutant protease TTEV and the TEVMP7 protease of the present invention are shown. DETAILED DESCRIPTION

[0029] The present invention provides a recombinant TEV protease. The amino acid sequence of the recombinant TEV protease is GASLFKGPRDYNPISSSICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLVVQSLHGVFKVKDTTTLQQHLVDGRDMIIIRMPKDFPPFPQKLKFREPQRGERICLVTTNFQTKSMSSMVSDTSCTFPSGDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKP (SEQ ID NO: 1). In the present invention, compared with the original TEV protease, the recombinant TEV protease has the 17th amino acid residue mutated from threonine to serine, the 56th amino acid residue mutated from leucine to valine, the 68th amino acid residue mutated from asparagine to aspartic acid, the 77th amino acid residue mutated from isoleucine to valine, the 106th amino acid residue mutated from glutamic acid to glycine, the 135th amino acid residue mutated from serine to glycine, and the 219th amino acid residue mutated from serine to valine. The present invention names the recombinant TEV protease TEVMP7, and the seven mutation sites of the TEVMP7 reduce the rate of its intramolecular autohydrolysis and enhance the stability of the protein structure. The design of the present invention prolongs the effective life of the protein and improves its stability under various application conditions.

[0030] The present invention also provides a gene encoding the recombinant TEV protease, the nucleotide sequence of the gene encoding the recombinant TEV protease is (SEQ ID NO: 2). The gene encoding the recombinant TEV protease of the present invention is synthesized by a gene synthesis company after codon optimization.

[0031] The present invention also provides a recombinant vector for expressing TEV protease, the recombinant vector comprising an initial vector and a coding gene for the recombinant TEV protease. In the present invention, the initial vector is preferably PET21M-SUMO3, which contains a SUMO tag. The present invention significantly improves the purity of the recombinant TEV protease by introducing a SUMO fusion tag. The SUMO tag can enhance the solubility of the target protein and reduce the risk of forming insoluble aggregates during the expression process. In addition, the SUMO fusion tag provides additional selectivity during the purification process, improving the separation effect of the target protein from impurities. In the present invention, the expression elements of the PET21M-SUMO3 include: the order is T7 promoter → 6XHIS tag → SUMO3 gene → restriction site → T7 terminator. The present invention preferably inserts the coding gene of the recombinant TEV protease between the two restriction sites of BamH I / XhoI of the initial vector, and the initial vector can be purchased through conventional channels. The PET21M-SUMO3 described in the present invention is synthesized by Shanghai Saiheng Biotechnology Co., Ltd.

[0032] The present invention also provides a method for expressing and purifying the recombinant TEV protease, comprising the following steps: transfecting the recombinant vector into Escherichia coli, selecting positive clones in the transfected Escherichia coli and inducing expression in a culture medium; collecting the bacterial cells by centrifugation, and lysing the collected bacterial cells with a resuspension buffer to obtain a lysate; centrifuging the lysate to collect the supernatant; and purifying the supernatant to obtain the recombinant TEV protease.

[0033] In the present invention, the Escherichia coli is preferably BL21-CodonPlus(DE3)-RIPL. The Escherichia coli (E. coli) selected in the present invention has the advantages of rapid proliferation, easy operation, low cost, etc., and is used for the expression of recombinant TEV protease to achieve efficient induced expression of the target protease. The Escherichia coli BL21-CodonPlus(DE3)-RIPL can be purchased through conventional channels. In the present invention, the Escherichia coli BL21-CodonPlus(DE3)-RIPL was purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0034] In the present invention, the coding gene of the recombinant TEV protease synthesized by the company is inserted into the initial vector PET21M-SUMO3 to obtain the recombinant vector PET21M-SUMO3-TEVMP7, and the recombinant vector PET21M-SUMO3-TEVMP7 is transfected into the competent state of Escherichia coli BL21-CodonPlus (DE3)-RIPL for large-scale induced expression. The induction expression step is preferably as follows: pick a single colony and inoculate it in LB culture medium, and culture it overnight at 37°C and 220rpm; take out the bacterial solution from the overnight culture medium and add it to the culture medium to ensure that the final concentration is 1% (v / v); then culture it on a shaker at 220rpm for 3 to 4 hours until the bacterial solution in the culture medium is visibly turbid, at which time the OD 600 Usually at 0.6-0.8; finally, the temperature is lowered to 22-25°C, IPTG is added at a final concentration of 0.1 mM, and the expression is induced for 15-18 hours. In the present invention, the LB medium preferably contains 100 μg / mL ampicillin. In the present invention, the induction expression condition is more preferably induced at 25°C for 16 hours.

[0035] In the present invention, after the induction of expression is completed, the bacteria are collected by centrifugation, and the resuspension buffer is used for homogenizer lysis to obtain a lysate; the lysate is collected and the supernatant is centrifuged. In the present invention, the resuspension buffer preferably includes 10mM Tris-HCl, 500mM NaCl; pH 7.4. In the present invention, the lysis condition is preferably lysis at a pressure of 600-1000Pa for 8-15min; more preferably lysis at a pressure of 800Pa for 10min. In the present invention, the centrifugation condition is preferably centrifuged at 20000-30000 rpm for 40-50min; more preferably centrifuged at 25000 rpm for 45min.

[0036] In the present invention, the supernatant is preferably purified by a Ni-NTA affinity column to obtain recombinant TEV protease. The specific steps are preferably as follows: an affinity column is connected with an ACHROM BASE (Taidu Biotechnology (Suzhou) Co., Ltd., hereinafter referred to as "Taidu Biotechnology") protein purifier, and the sample of the supernatant is loaded with a Ni-NTA affinity column at a flow rate of 5 mL / min, and non-specific proteins are removed with a washing buffer containing 20 mM imidazole (10 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.4), and non-specific proteins are further removed with a washing buffer containing 60 mM imidazole (10 mM Tris-HCl, 500 mM NaCl, 60 mM imidazole, pH 7.4); the target protein is eluted with an elution buffer of 200 mM imidazole. In the present invention, the elution process preferably adopts isocratic elution.

[0037] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0039] Example 1

[0040] A recombinant TEV protease, named TEVMP7. The amino acid sequence of the recombinant TEV protease is SEQ ID NO: 1, and the nucleotide sequence encoding the recombinant TEV protease is SEQ ID NO: 2. Compared with the original TEV protease (wild-type protease TEV), the amino acid residue at position 17 of the recombinant TEV protease is mutated from threonine to serine, the amino acid residue at position 56 is mutated from leucine to valine, the amino acid residue at position 68 is mutated from asparagine to aspartic acid, the amino acid residue at position 77 is mutated from isoleucine to valine, the amino acid residue at position 106 is mutated from glutamic acid to glycine, the amino acid residue at position 135 is mutated from serine to glycine, and the amino acid residue at position 219 is mutated from serine to valine.

[0041] The gene sequence of the recombinant TEV protease was synthesized by Shanghai Saiheng Biotechnology Co., Ltd.

[0042] Example 2

[0043] A method for expressing and purifying recombinant TEV protease, the specific steps are as follows:

[0044] 1. Plasmid transfection:

[0045] The gene sequence of the recombinant TEV protease synthesized in Example 1 was inserted between the two restriction sites of Bam HI / Xho I of plasmid PET21M-SUMO3 to obtain recombinant plasmid PET21M-SUMO3-TEVMP7, and the recombinant plasmid PET21M-SUMO3-TEVMP7 was transfected into Escherichia coli BL21-CodonPlus (DE3)-RIPL competent cells. The map of plasmid PET21M-SUMO3 is shown in Figure 1 The map of recombinant plasmid PET21M-SUMO3-TEVMP7 is shown in Figure 2 .

[0046] 2. Bacteria culture:

[0047] Pick a single colony and inoculate it into 130ml LB medium, and culture it in a shaker at 37℃ and 220rpm overnight; take out 10mL of bacterial solution from the overnight culture medium and add it to 1L of culture medium to ensure that the final concentration is 1% (v / v), a total of 1L; culture it in a shaker at 220rpm for 3-4 hours until the bacterial solution in the culture medium is visibly turbid, at which time OD 600 Usually at 0.6-0.8; lower the temperature to 25°C, add IPTG with a final concentration of 0.1mM, and induce expression for 16 hours.

[0048] 3. Bacteria collection and lysis:

[0049] The bacterial solution after induction expression in step 2 was centrifuged to collect the bacteria, and lysed for 10 min at 800 Pa pressure in a homogenizer using a resuspension buffer (10 mM Tris-HCl, 500 mM NaCl, pH 7.4); the lysate was collected and centrifuged at 25,000 rpm for 45 min to obtain 50 mL of the supernatant.

[0050] 4. Affinity chromatography purification:

[0051] The affinity column was connected with ACHROM BASE protein purifier (Taidu Biotechnology), and 50 mL of the supernatant sample obtained in step 3 was loaded onto the Ni-NTA affinity column at a flow rate of 5 mL / min. Non-specific proteins were removed with a washing buffer containing 20 mM imidazole (10 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.4), and then non-specific proteins were further removed with a washing buffer containing 60 mM imidazole (10 mM Tris-HCl, 500 mM NaCl, 60 mM imidazole, pH 7.4); finally, the target protein was isocratically eluted using an elution buffer of 200 mM Imidazole to obtain the recombinant TEV protease.

[0052] Example 3

[0053] In this example, SDS-PAGE electrophoresis, ACHROM BASE protein purifier (Taidu Biotechnology) software Chiron, and Image J software were used to detect the expression results of the recombinant TEV protease (TEVMP7) obtained in Example 2. The SDS-PAGE kit was purchased from Nanjing GenScript Biotechnology Co., Ltd., the dye solution was a product of Zhejiang Yunan Kangrui Biotechnology Co., Ltd., the ACHROM BASE protein purifier (Taidu Biotechnology) software Chiron was built into the device, and the Image J software was downloaded from the Internet, version 1.51j8.

[0054] The samples and amounts for SDS-PAGE loading in this example are as follows:

[0055] ①Supernatant after TEVMP7 lysis ( Figure 3 The supernatant of the second lane in the sample was loaded with 15 μL of sample diluted 20 times;

[0056] ② The supernatant after lysate is passed through the NTA affinity column and then the flow-through ( Figure 3 FT of the third lane in the sample), sample volume: dilute the sample 20 times and load 15 μL;

[0057] ③ Wash with 20mM imidazole buffer ( Figure 3 AW1 in lane 4), sample volume: 15 μL;

[0058] ④ Wash with 60mM imidazole buffer ( Figure 3 AW2 in lane 5), sample volume: 15 μL;

[0059] ⑤ Target protein sample (recombinant TEV protease) after elution with 200mM imidazole ( Figure 3 Lanes 6-11 in the figure), sample volume: 5 μL;

[0060] according to Figure 3 The results of SDS-PAGE electrophoresis showed that the target protein was about 40kda and was expressed.

[0061] This example uses the ACHROM BASE protein purifier (Taidu Biotechnology) software Chiron to operate as follows:

[0062] ① Select the collected peaks and click Peak Area Integration Calculation ( Figure 4 is the peak to be calculated by box selection);

[0063] ② Calculate and obtain the peak area as 15435.30mAU*ml;

[0064] ③The formula is as follows:

[0065]

[0066] Where m is mass in mg. c is concentration in mg / mL. A is absorbance in Au. ε is the absorption coefficient of protein. l is the optical path length of the device in cm. V is volume in mL.

[0067] ④ The peak area A×V is known to be 15435.30mAU*ml, i.e. 15.4Au*ml, ε is 1 from the protein sequence, and the optical path length of this device is 0.2cm. Substituting this into the calculation, m is 77mg.

[0068] The Image J software of this embodiment is operated as follows:

[0069] ① Select the part whose purity needs to be measured ( Figure 5The yellow frame is the selected part);

[0070] ②The software will automatically scan and calculate the brightness of the selected part from top to bottom.

[0071] ③The software will give the peak area corresponding to different brightness, and finally give the proportion of each peak to the total peak. The proportion is the purity. The purity obtained by the software is 97%.

[0072] ④Finally, the final target protein yield was calculated, 77 mg × 97% = 75 mg. Since this protein has a SUMO3 tag, its molecular weight ratio is 3 (SUMO3 tag): 5 (TEVMP7 protein), so the final pure TEVMP7 protein (without SUMO tag) is about 45 mg.

[0073] according to Figures 3 to 5 The results showed that the purity of TEVMP7 was 97%, 45 mg of protein sample was collected from 1 L of E. coli, and the expression yield was about 45 mg / L.

[0074] Comparative Example 1

[0075] TEV (wild-type protease) was expressed and purified according to the methods of Examples 1 to 3.

[0076] SDS-PAGE results: Target protein sample (recombinant TEV protease) after elution with 200 mM imidazole ( Figure 6 Lanes 6-12 in the figure), sample volume: 5 μL;

[0077] ACHROM BASE protein purifier (Taidu Biotechnology) was used with Chiron software. Figure 7 )Integral calculation result: 4048.46mAU*ml. TEV (wild-type protease) ε value is 1.22, and the calculated sample protein amount is about 17mg;

[0078] Image J software calculation operation ( Figure 8 ), the software obtained a purity of 92%, 17mg×92%=16mg, and the final yield was about 15mg / L. TEV (wild-type protease) will undergo autohydrolysis ( Figure 6 6-12 in the swimming lanes), it can be observed that TEV (wild-type protease) is divided into two bands. Under the same conditions, TEVMP7 does not undergo autolysis ( Figure 3 Lanes 6-11).

[0079] By comparison: TEVMP7 yield 45mg / L, purity 97%; TEV (wild-type protease) yield 15mg / L, purity 92%. The results show that the yield and purity of TEVMP7 mutant protease are higher than TEV (wild-type protease).

[0080] Example 4

[0081] In this example, the activity of TEV enzymes of TEV (wild-type protease), TTEV (single mutant protease), and TEVMP7 (mutant protease of Example 2) was measured, and the substrate for enzyme cleavage was S-GST. Among them, compared with the wild-type protease TEV, the 219th amino acid residue of TTEV (single mutant protease) mutated from serine to valine. The amount of 30 μg substrate (S-GST) that can be cut by different genotype proteins at the same TEV protein concentration and reacted at 30°C for 60 minutes was measured. The total reaction system was 100 μL and tested in 4 groups:

[0082] Group 1: 3 centrifuge tubes, each with 30 μg of substrate (S-GST) and about 30 μg of enzymes TEV, TTEV, and TEVMP7, to observe the efficiency of enzyme cleavage.

[0083] Group 2: 3 centrifuge tubes, each with 30 μg of substrate (S-GST), about 30 μg of enzymes TEV and TTEV, and about 15 μg of enzyme TEVMP7, to observe the enzyme cleavage efficiency of the reorganization.

[0084] Group 3: control group, 3 centrifuge tubes, no substrate was added, and about 30 μg of enzyme TEV, TTEV, and about 15 μg of enzyme TEVMP7 were added to each tube.

[0085] Group 4: 24-hour enzyme digestion control at room temperature. 20 μg of substrate (S-GST) and about 10 μg of enzymes TEV, TTEV, and TEVMP7 were added to three centrifuge tubes to observe the efficiency of enzyme digestion.

[0086] The loading volume is 1 / 10, i.e. 10 μL. Therefore, the content of each sample is reduced to 1 / 10.

[0087] The samples and results of SDS-PAGE loading in this example are as follows:

[0088] The red boxes indicate the positions of the proteases, and the substrate S-GST is the protein at 55-70 kda in lane 2.

[0089] Lane 1: Maker.

[0090] Lane 2: S-GST, loading amount: 3 μg.

[0091] Lanes 3-5: TEV 3μg+S-GST 3μg, TTEV 3μg+S-GST 3μg, TEVMP73μg+S-GST 3μg. S-GST was cleaved to generate a 40kda protein, which is particularly evident in lane 5.

[0092] Lane 6: S-GST, loading amount 3 μg.

[0093] Lanes 7-9, TEV 3μg + S-GST 3μg, TTEV 3μg + S-GST 3μg, TEVMP71.5μg + S-GST3μg. Similarly, S-GST was cleaved to generate a protein of 40kda, which is particularly obvious in lane 5.

[0094] Lanes 10-12: TEV 3 μg, TTEV 3 μg, TEVMP7 1.5 μg, respectively.

[0095] Lanes 13-15: 24 hours of enzyme digestion, TEV 1μg + S-GST 2μg, TTEV 1μg + S-GST 2μg, TEVMP71μg + S-GST 2μg. S-GST was cleaved by enzymes, TEV and TTEV cleaved half of the substrate S-GST, while TEVMP7 cleaved almost all of the substrate.

[0096] The results are as follows Fig. 9 As shown: Under the same conditions, TEVMP7 has the highest activity among TEV, TTEV and TEVMP7 with the same concentration. When 30μg TEVMP7 is reacted at 30℃ for 60 minutes, 3μg of substrate (S-GST) can be clearly observed to be cleaved by enzyme, while the activity of TEV and TTEV is relatively unobvious.

[0097] Under the same conditions at room temperature, with an enzyme: substrate ratio of 1:2, TEVMP7 can cleave all substrates within 24 hours, while TEV and TTEV can only cleave half of the substrates.

[0098] In summary, the 7-mutant (TEVMP7) TEV enzyme obtained by the present invention has the highest activity, and the activity is much higher than that of the wild type. In addition, the SUMO tag of the TEVMP7 protease is not cleaved (40 kDa), and it can still play a normal catalytic role.

[0099] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant TEV protease, characterized in that The amino acid sequence of the recombinant TEV protease is shown in SEQ ID NO:

1.

2. The recombinant TEV protease according to claim 1, characterized in that Compared with the original TEV protease, the recombinant TEV protease has the following characteristics: the 17th amino acid residue of the recombinant TEV protease is mutated from threonine to serine, the 56th amino acid residue of the recombinant TEV protease is mutated from leucine to valine, the 68th amino acid residue of the recombinant TEV protease is mutated from asparagine to aspartic acid, the 77th amino acid residue of the recombinant TEV protease is mutated from isoleucine to valine, the 106th amino acid residue of the recombinant TEV protease is mutated from glutamic acid to glycine, the 135th amino acid residue of the recombinant TEV protease is mutated from serine to glycine, and the 219th amino acid residue of the recombinant TEV protease is mutated from serine to valine.

3. A gene encoding the recombinant TEV protease according to claim 1 or 2, characterized in that: The nucleotide sequence of the gene encoding the recombinant TEV protease is shown in SEQ ID NO:

2.

4. A recombinant vector expressing TEV protease, characterized in that: The recombinant vector comprises an initial vector and the gene encoding the recombinant TEV protease according to claim 3.

5. The recombinant vector according to claim 4, characterized in that The initial vector is PET21M-SUMO3.

6. A method for expressing and purifying a recombinant TEV protease, characterized in that: The method comprises the following steps: transfecting the recombinant vector according to claim 4 or 5 into Escherichia coli, selecting positive clones in the transfected Escherichia coli and inducing expression in a culture medium; collecting the bacteria by centrifugation, and lysing the collected bacteria with a resuspension buffer to obtain a lysate; centrifuging the lysate to collect the supernatant; and purifying the supernatant to obtain the recombinant TEV protease.

7. The expression purification method according to claim 6, characterized in that: The Escherichia coli is BL21-CodonPlus (DE3)-RIPL.

8. The expression purification method according to claim 6, characterized in that: The culture medium is selected from LB culture medium containing 100 μg / mL ampicillin.

9. The expression purification method according to claim 6, characterized in that: The induction expression condition is 22-25° C. for 15-18 hours.

10. The expression purification method according to claim 6, characterized in that: The supernatant was purified using a Ni-NTA affinity column.

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