Recombinant TEV protease, preparation method therefor, and use thereof

Abstract

A recombinant TEV protease, a preparation method therefor, and use thereof, relating to the technical field of biology. Specifically provided are four recombinant TEV proteases derived from a cysteine protease of tobacco etch virus. In each of the four recombinant TEV proteases, the N-terminus is fused with an 8His-CL7-GGS tag, and the C-terminus is fused with a polyarginine tag. Compared to a wild-type TEV protease, the provided four recombinant TEV proteases not only can overcome defects such as self-cleavage, poor solubility, and low activity during expression and purification of the wild-type TEV protease, but also have improved protein yield 5-6 times higher than that of the wild-type TEV enzyme, and enzyme activity 10-20 times higher than that of the wild-type TEV protease. In addition, the engineered TEV protease mutants also possess higher thermal stability with a Tm value 10-30 °C higher than that of the wild-type TEV protease, exhibit better protein stability, have broader application conditions, and are more suitable for large-scale production and industrial use.

Description

A recombinant TEV protease, its preparation method and application Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a recombinant TEV protease, its preparation method, and its applications. Background Technology

[0002] TEV protease is a cysteine ​​protease derived from Tobacco Etch Virus (TEV). It exhibits strong site specificity, strictly recognizing the heptapeptide sequence E-Xaa-Xaa-Y-Xaa-QG / S (the commonly used heptapeptide sequence is ENLYFQG), with the cleavage site located between the last two amino acid residues. This ensures that only one amino acid residue remains at the N-terminus of the target protein after cleavage, minimizing the impact on the protein's structure and function. Furthermore, TEV protease possesses broad pH and temperature tolerance, tolerating pH levels from 4 to 8.0 and temperatures from 4 to 34°C. It also exhibits varying degrees of tolerance to common additives that increase protein solubility or stability (ethylene glycol, EGTA, detergents, and reducing agents). Therefore, TEV protease is an ideal tool enzyme for removing fusion tags from recombinant proteins.

[0003] However, wild-type TEV proteases suffer from defects such as self-splicing, rare codons, and poor solubility during expression and purification in *E. coli*. Numerous reports have addressed these defects by modifying TEV proteases, for example, by adding fusion MBP tags or short polypeptide sequences, or by designing TEV protease mutants through directed evolution. While these modifications help improve the solubility and expression levels of TEV proteases, they have not significantly enhanced enzyme activity.

[0004] Patent application CN202111024156.X discloses a recombinant TEV enzyme with six mutation sites (T17S, L56V, N68D, I77V, S135G, S219N), an N-terminal fused with a CL7 tag, and a C-terminal fused with a polyarginine tag, enabling efficient expression and exhibiting high activity and stability. Based on the prior patent application CN202111024156.X, this invention further modifies the TEV protease, optimizing the number of amino acid mutations, and provides four modified TEV proteases, their preparation methods, and applications. These four modified TEV proteases exhibit higher yield, activity, and stability, making them suitable for a wider range of applications and large-scale production and industrial use.

[0005] Summary of the Invention

[0006] The purpose of this invention is to provide a recombinant TEV protease, its preparation method, and its application in order to further improve the yield, activity, and stability of TEV protease.

[0007] The present invention achieves the above objectives through the following technical solutions:

[0008] As a first aspect of the present invention, a recombinant TEV protease is provided, the recombinant TEV protease being BioTEVp-1, BioTEVp-2, BioTEVp-3 and BioTEVp-4, the amino acid sequences of BioTEVp-1, BioTEVp-2, BioTEVp-3 and BioTEVp-4 being as shown in SEQ ID NO.1-4, respectively.

[0009] As a further optimization of the present invention, the recombinant TEV protease has an 8His-CL7-GGS tag attached to its N-terminus and a polyarginine tag attached to its C-terminus.

[0010] As a further optimization of the present invention, the sequence of the 8His-CL7-GGS tag is as shown in SEQ ID NO.7, and the sequence of the polyarginine tag is RRRGRRRGRRRG.

[0011] As a second aspect of the present invention, a polynucleotide is also provided, the polynucleotide encoding the recombinant TEV protease as described above, and the polynucleotide sequences encoding BioTEVp-1, BioTEVp-2, BioTEVp-3 and BioTEVp-4 are shown in SEQ ID NO. 8-11, respectively.

[0012] As a third aspect of the present invention, a recombinant plasmid is also provided, the recombinant plasmid being an expression vector containing any of the polynucleotide sequences described above and capable of correspondingly translating and expressing any of the recombinant TEV proteases described above, the expression vector being pET-28a.

[0013] As a fourth aspect of the invention, a recombinant TEV protease expression system is also provided, comprising BL21(DE3) cells containing any of the recombinant plasmids described above or whose genomes integrate any of the polynucleotides described above.

[0014] As a fifth aspect of the present invention, a method for preparing recombinant TEV protease as described in any of the above-described steps is also provided, comprising the following steps:

[0015] (1) Based on the amino acid sequence of the recombinant TEV protease, a nucleotide sequence that can encode the amino acid sequence is synthesized, and its gene is constructed in the pET-28a vector to obtain a recombinant plasmid.

[0016] (2) The recombinant plasmid was expressed using the Escherichia coli prokaryotic expression system and the modified recombinant TEV protease mutant was obtained by purification using Ni-NTA column and affinity chromatography.

[0017] As a sixth aspect of the invention, the application of the recombinant TEV protease as described in any of the above-described methods as a proteolytic enzyme in protein purification is also provided.

[0018] As a seventh aspect of the present invention, a method for determining the activity of the recombinant TEV protease as described above is also provided, namely, using a fluorescence resonance energy transfer method to detect the activity of the TEV protease.

[0019] As an eighth aspect of the present invention, a method for determining the thermal stability of recombinant TEV protein as described above is also provided, wherein the stability of the protein is determined by measuring the change in the dissolution temperature Tm value of the mutant protein; the higher the Tm value, the more stable the protein.

[0020] The present invention has the following beneficial effects:

[0021] The four modified recombinant TEV protease mutants, BioTEVp-1, BioTEVp-2, BioTEVp-3, and BioTEVp-4, provided by this invention possess high yield, activity, and thermostability, specifically manifested in:

[0022] (1) In terms of protein yield, under the same conditions, BioTEVp-1 protein yield is as high as 128.4 mg / L, BioTEVp-2 protein yield is as high as 122.85 mg / L, BioTEVp-3 protein yield is as high as 119.7 mg / L, and BioTEVp-4 protein yield is as high as 107.55 mg / L. Compared with the existing TEVp-control 1, the yield is nearly 2 times higher, and compared with TEVp-control 2, it is nearly 5-6 times higher.

[0023] (2) Regarding protein activity, the activity of BioTEVp-1 was 0.040 (RFU / s / nM), the activity of BioTEVp-2 was 0.021 (RFU / s / nM), the activity of BioTEVp-3 was 0.019 (RFU / s / nM), and the activity of BioTEVp-4 was 0.039 (RFU / s / nM). The activity of BioTEVp-1 was nearly 4 times higher than that of TEVp-control 1 and nearly 20 times higher than that of TEVp-control 1. The activities of BioTEVp-2 and BioTEVp-3 were nearly 2 times higher than that of TEVp-control 1 and nearly 10 times higher than that of TEVp-control 2.

[0024] (3) In terms of thermal stability, the Tm value of BioTEVp-1 is 81.25℃, the Tm value of BioTEVp-2 is 83.26℃, the Tm value of BioTEVp-3 is 75.75℃, and the Tm value of BioTEVp-4 is 58.55℃. The thermal stability of BioTEVp-1, BioTEVp-2, and BioTEVp-3 is significantly improved, increasing by 24.18℃, 26.19℃, 18.68℃, and 1.48℃ compared to TEVp-Control 1, and by 31.97℃, 33.98℃, 26.47℃, and 9.27℃ compared to TEVp-Control 2. Among them, the Tm values ​​of BioTEVp-1 and BioTEVp-2 are as high as 80℃ or more.

[0025] In summary, compared with existing recombinant TEV proteases and wild-type TEV proteases, the high yield, high activity, and high thermal stability of the four modified recombinant TEV proteases BioTEVp-1, BioTEVp-2, BioTEVp-3, and BioTEVp-4 make them suitable for a wider range of applications and large-scale industrial use. Attached Figure Description

[0026] Figure 1 shows the pET-28a expression vector containing the tag and polypeptide sequence;

[0027] Figure 2 shows the pET-28a expression vector containing the 6His tag;

[0028] Figure 3 shows the results of low-level expression detection of the modified TEV protease and the control TEV protease;

[0029] Figure 4 shows the affinity purification results of the modified TEV protease and the control TEV protease;

[0030] Figure 5 shows the activity detection results of the modified TEV protease and the control TEV protease;

[0031] Figure 6 shows the thermostability results of the modified TEV protease and the control TEV protease.

[0032] Figure 7 shows the results of the enzymatic digestion application of the modified TEV protease. Detailed Implementation

[0033] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0034] 1. Materials and Reagents

[0035] Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art. Where specific conditions are not specified, they shall be performed according to conventional conditions or conditions recommended by the manufacturer. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0036] 2. Method

[0037] 2.1 Construction of recombinant TEV protease plasmid

[0038] The gene sequences of the recombinant TEV proteases provided in this invention were all obtained through gene synthesis. The four mutant TEV proteases are BioTEVp-1 (amino acid sequence shown in SEQ ID NO. 1), BioTEVp-2 (amino acid sequence shown in SEQ ID NO. 2), BioTEVp-3 (amino acid sequence shown in SEQ ID NO. 3), and BioTEVp-4 (amino acid sequence shown in SEQ ID NO. 4), with corresponding gene sequences shown in SEQ ID NO. 8-11. The synthesized genes of the four mutant TEV proteases were constructed on the expression vector pET-28a (the pET-28a expression vector containing the tag and polypeptide sequence is shown in Figure 1). The N-terminus carries the tag 8His-CL7-GGS (the amino acid sequence of the tag is shown in SEQ ID NO. 7; in the sequence, positions 1-8 are the 8His tag, positions 9-138 are the CL7 tag, and positions 139-141 are the GGS), and the C-terminus carries the polypeptide sequence RRRGRRRGRRRG, used to improve protein solubility and facilitate purification.

[0039] TEV protease used as a control:

[0040] TEVp-Control 1 (amino acid sequence see SEQ ID NO.5) is the recombinant TEV protease in patent CN202111024156.X. After its gene is synthesized, it is constructed on the expression vector pET-28a. The N-terminal and C-terminal tag or polypeptide sequences are consistent with those of the four mutant TEV proteases.

[0041] TEVp-control 2 (amino acid sequence see SEQ ID NO.6) has only one amino acid sequence where serine is mutated to aspartic acid at position 219, which is the wild-type TEV protease. After its gene is synthesized, it is constructed on the expression vector pET-28a (the map of the expression vector pET-28a containing the 6His tag is shown in Figure 2). The N-terminus has the 6His tag, and the sequence is HHHHHH.

[0042] All recombinant plasmids were sequenced and verified to be completely identical to the target sequence.

[0043] 2.2 Expression of recombinant TEV protease protein

[0044] 2.2.1 Low-level expression of recombinant TEV protease

[0045] Using standard molecular biology techniques, the constructed recombinant TEV protease plasmid was transformed into BL21(DE3) *E. coli* competent cells in a clean bench and cultured overnight at 37°C. Single colonies from the overnight culture were picked and transferred to 5 ml of LB broth and incubated at 37°C until the bacterial culture showed an OD value. 600 When the pH is 0.6-0.8, a small amount of bacterial culture is fixed with loading buffer, and a small amount of bacterial culture is added to glycerol and frozen to -80℃. The remaining bacterial culture is added to 0.5mM IPTG and induced at 15℃ for 16 hours. The bacterial cells are then collected and the induced bacterial culture is analyzed by SDS-PAGE.

[0046] The results are shown in Figure 3. Compared with TEVp-control 1 and TEVp-control 2, the target bands of BioTEVp-1, BioTEVp-2, BioTEVp-3 and BioTEVp-4 proteases were obvious, and they were all clearly expressed in BL21(DE3) Escherichia coli and had good protein solubility.

[0047] 2.2.2 High-level expression of recombinant TEV protease

[0048] The strains exhibiting the above-mentioned clear expression were inoculated into 50 ml of LB liquid medium and cultured overnight at 37°C. The bacteria cultured overnight were then inoculated into 1 L of LB liquid medium at a ratio of 1:100 and cultured at 37°C until the bacterial culture reached OD. 600 When the bacterial growth rate is 0.6-0.8, add 0.5mM IPTG and incubate overnight at 15℃. Collect the bacterial cells by centrifugation at 5000rpm.

[0049] 2.3 Purification of recombinant TEV protease

[0050] 2.3.1 Affinity chromatography of recombinant TEV protease

[0051] The collected bacterial cells were weighed and added to a lysis buffer (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5% glycerol) at a 1:10 ratio. The cells were then homogenized using a high-pressure homogenizer, and the supernatant was collected by centrifugation at 16,000 rpm. All recombinant TEV proteases were His-tagged, and the proteins were enriched and purified using a Ni Bestarose FF affinity chromatography column. The specific procedure was as follows: the Ni Bestarose FF affinity chromatography column was first equilibrated with lysis buffer to 10 column volumes. Then, the lysis supernatant was loaded onto the Ni Bestarose FF affinity chromatography column, and eluted with imidazole solutions of different gradients. The proteins eluted with different gradients of imidazole were collected for SDS-PAGE analysis, and the protein concentration was determined using Nanodrop to calculate the protein yield.

[0052] The protein purification results are shown in Figure 4. Four mutant recombinant TEV proteases and two control TEV proteases with high purity were obtained. The purification results show that BioTEVp-1, BioTEVp-2, BioTEVp-3, and BioTEVp-4 proteases were produced in higher quantities in BL21(DE3) cells than the existing TEVp-control 1 and TEVp-control 2 proteases. Nanodrop assays showed that the protein yield of BioTEVp-1 was 128.4 mg / L; BioTEVp-2 was 122.85 mg / L; BioTEVp-3 was 119.7 mg / L; BioTEVp-4 was 107.55 mg / L; TEVp-control 1 was 63 mg / L; and TEVp-control 2 was 21.6 mg / L. Comparing yields, it can be seen that the yields of BioTEVp-1, BioTEVp-2, BioTEVp-3, and BioTEVp-4 proteases were nearly 2 times higher than those of TEVp-control 1 and nearly 5-6 times higher than those of TEVp-control 2, showing a significant increase in yield.

[0053] 2.4 Activity test of recombinant TEV protease

[0054] The activity of the TEV protease was detected using fluorescence resonance energy transfer (FRET), specifically, in the presence of two different fluorescent groups, the emission spectrum of one fluorescent group (donor) overlaps to some extent with the absorption spectrum of the other group (acceptor), and the distance between the two fluorescent groups is less than [missing value]. Fluorescent energy is transferred from the donor to the acceptor, resulting in a significantly lower fluorescence intensity in the donor compared to its own (fluorescence quenching). FAM and TAMRA fluorescent groups are attached to both ends of the polypeptide sequence containing the specific recognition site. When the polypeptide is cleaved, the two fluorescent groups separate, releasing a strong fluorescent signal. The higher the activity of the TEV protease, the more fluorescent groups are released, and the greater the fluorescence intensity. Enzyme activity is expressed as the fluorescence intensity absorbed per nanomolar of protein per second.

[0055] The specific procedure for TEV protease activity assay is as follows:

[0056] The peptide substrate (ENLYFQGSG labeled with FAM at the 5' end and TAMRA at the 3' end) was prepared into a 1 mM stock solution and aliquoted for use. The TEV protease activity assay buffer consisted of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM DTT, and 0.5 mM EDTA. The peptide concentration was diluted to 100 nM with the buffer, and the TEV protease was serially diluted 2-fold from 1 μM to a total of 12 concentrations. 30 μL of the substrate was transferred to a 384-well plate with two replicates. 30 μL of the TEV protease to be tested was transferred to the corresponding well, and the plates were immediately centrifuged and vortexed to mix. The fluorescence signal values ​​generated by the reaction were collected using a TECANF200 microplate reader. Data analysis was performed using Graph Pad Prism9 software to obtain the enzyme activity parameters of the test protease. Figure 5 shows the TEV protease activity parameters obtained by Graph Pad Prism9 software analysis.

[0057] The activity assays for BioTEVp-1 and BioTEVp-4 were 0.040 (RFU / s / nM), 0.021 (RFU / s / nM), 0.019 (RFU / s / nM), 0.039 (RFU / s / nM), 0.011 (RFU / s / nM) for TEVp-Control 1, and 0.002 (RFU / s / nM) for TEVp-Control 2. The activities of BioTEVp-1 and BioTEVp-4 were nearly 4 times higher than those of TEVp-Control 1 and nearly 20 times higher than those of TEVp-Control 2; the activities of BioTEVp-2 and BioTEVp-3 were nearly 2 times higher than those of TEVp-Control 1 and nearly 10 times higher than those of TEVp-Control 2. Therefore, the enzyme activities of BioTEVp-1, BioTEVp-2, BioTEVp-3, and BioTEVp-4 were significantly increased, with BioTEVp-1 and BioTEVp-4 showing an activity increase of nearly 20 times. These results indicate that the TEV protease mutants described in this invention exhibit higher activity compared to existing TEV protease mutants.

[0058] 2.5 Thermal stability test of recombinant TEV protease

[0059] The thermal stability of recombinant TEV protease was tested using protein thermal shift (ThermoFluor) technology. This technique utilizes the protein's structural characteristics; proteins possess hydrophobic regions hidden internally. As temperature rises, this structure opens up, exposing the hydrophobic regions. The fluorescent dye SYPRO Orange can then bind to these regions, stimulating fluorescence. A melting curve is formed based on the change in fluorescence intensity. The temperature corresponding to the maximum derivative of the melting curve is the melting point (Tm). The more stable the protein, the higher the measured Tm value.

[0060] The specific procedure for determining the thermal stability of TEV protease is as follows:

[0061] Add 5 μg of TEV protease to each well of a 96-well PCR plate, and then add 10×SYPRO Orange fluorescent dye to the corresponding wells. Place the 96-well PCR plate in a qPCR instrument, set the instrument parameters, and increase the temperature from 25℃ to 99℃ at a gradient of 1℃ per minute. Calculate the protein melting curve.

[0062] The results are shown in Figure 6. The Tm value of BioTEVp-1 was 81.25℃, the Tm value of BioTEVp-2 was 83.26℃, the Tm value of BioTEVp-3 was 75.75℃, the Tm value of BioTEVp-4 was 58.55℃, the Tm value of TEVp-Control 1 was 57.07℃, and the Tm value of TEVp-Control 2 was 49.28℃. The thermostability of BioTEVp-4 was similar to that of TEVp-control 1, increasing by 1.48℃, but 9.27℃ higher than TEVp-control 2. The thermostability of BioTEVp-1, BioTEVp-2, and BioTEVp-3 was significantly improved, increasing by 24.18℃, 26.19℃, and 18.68℃ compared to TEVp-control 1, and by 31.97℃, 33.98℃, and 26.47℃ compared to TEVp-control 2, respectively. Notably, the Tm values ​​of BioTEVp-1 and BioTEVp-2 reached over 80℃. The high thermostability of the four modified TEV proteases—BioTEVp-1, BioTEVp-2, BioTEVp-3, and BioTEVp-4—overcomes the problem of enzyme instability at high temperatures that limits their application, providing better application scenarios for their storage and industrial production.

[0063] 2.6 Application of Enzyme Digestion Verification

[0064] To test the practical application effect of the modified TEV protease, the recombinant protein GST-TEV-Pro was used as the enzyme cleavage target. This recombinant protein has a TEV sequence recognized by the TEV protease at its N-terminus, so the TEV enzyme can cleave the recombinant protein from the TEV restriction site into two fragments, GST and Pro.

[0065] In this experiment, the reaction buffer consisted of 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 5% glycerol. The enzymatic digestion reaction was performed at 4°C. TEV protease and recombinant protein were incubated at a mass ratio of 1:40. Samples were collected at four different time points (0.5 h, 2 h, and overnight) for SDS-PAGE analysis to compare the digestion efficacy of BioTEVp-1, BioTEVp-2, BioTEVp-3, BioTEVp-4, and TEVp-control 2 on GST-TEV-Pro protein.

[0066] The results are shown in Figure 7. At 4℃, after 0.5 hours of enzyme digestion, approximately 50% of the recombinant GST-TEV-Pro protein was digested. BioTEVp-4 showed better digestion efficiency, while the other three BioTEVp enzymes showed comparable digestion efficiency. After overnight reaction, the GST-TEV-Pro protein was basically cleaved into two bands, indicating that BioTEVp has high digestion efficiency in practical applications, with BioTEVp-4 showing even higher digestion efficiency.

[0067] 3. Conclusion

[0068] The above description shows that the four modified recombinant TEV proteases BioTEVp-1, BioTEVp-2, BioTEVp-3, and BioTEVp-4 provided by the present invention have higher protein yield, higher enzyme activity, and better protein stability, have broader application conditions and stronger practical application value, and are more suitable for large-scale production and industrial use.

[0069] The sequence involved in this invention:

[0070] SEQ ID NO.1 BioTEVp-1 protein sequence

[0071] SEQ ID NO.2 BioTEVp-2 protein sequence

[0072] SEQ ID NO.3

[0073] SEQ ID NO.4 BioTEVp-4 protein sequence

[0074] SEQ ID NO.5 TEVp-Control 1

[0075] SEQ ID NO.6 TEVp-Control 2

[0076] SEQ ID NO.7 8His-CL7-GGS tag sequence

[0077] SEQ ID NO.8 BioTEVp-1 nucleotide sequence

[0078] SEQ ID NO.9 BioTEVp-2 nucleotide sequence

[0079] SEQ ID NO.10 BioTEVp-3 nucleotide sequence

[0080] Nucleotide sequence of SEQ ID NO.11 BioTEVp-4

[0081] SEQ ID NO.12 Polyarginine tag sequence

[0082] SEQ ID NO.13 6His tag sequence

[0083] SEQ ID NO.14

[0084] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A recombinant TEV protease, characterized in that, The recombinant TEV protease is BioTEVp-1, BioTEVp-2, BioTEVp-3 and BioTEVp-4, and the amino acid sequences of the BioTEVp-1, BioTEVp-2, BioTEVp-3 and BioTEVp-4 are shown in SEQ ID NO. 1-4, respectively.

2. The recombinant TEV protease according to claim 1, wherein, The N-terminal of the recombinant TEV protease is connected with an 8His-CL7-GGS tag, and the C-terminal is connected with a polyarginine tag.

3. The recombinant TEV protease according to claim 2, wherein, The sequence of the 8His-CL7-GGS tag is shown in SEQ ID NO. 7, and the sequence of the polyarginine tag is RRRGRRRGRRRG.

4. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of claim 1 or 2. The polynucleotide encodes the recombinant TEV protease of claim 1.

5. The polynucleotide of claim 4, wherein The polynucleotide sequences encoding BioTEVp-1, BioTEVp-2, BioTEVp-3 and BioTEVp-4 are shown in SEQ ID NO. 8-11, respectively.

6. A recombinant plasmid, characterized in that, The recombinant plasmid is an expression vector containing the polynucleotide sequence of any one of claims 4-5, and can correspondingly express the recombinant TEV protease of any one of claims 1-3.

7. The recombinant plasmid of claim 6, wherein, The expression vector is pET-28a.

8. An expression system for recombinant TEV protease, characterized in that, The BL21(DE3) cell containing the recombinant plasmid of any one of claims 6-7 or the polynucleotide of any one of claims 4-5 integrated in the genome.

9. A method of producing a recombinant TEV protease according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: (1) synthesizing a nucleotide sequence capable of encoding the amino acid sequence of the recombinant TEV protease according to the amino acid sequence, constructing the gene on a pET-28a vector to obtain a recombinant plasmid; (2) expressing the recombinant plasmid by using an E. coli prokaryotic expression system, and purifying the modified recombinant TEV protease mutant by using a Ni-NTA column and affinity chromatography.

10. Use of the recombinant TEV protease of any one of claims 1-3 as a proteolytic enzyme in a protein purification process.

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