CN-122012471-A - TEV protease variant and application thereof

Abstract

The invention relates to TEV protease, in particular to a TEV protease variant and application thereof, and provides a TEV protease mutant, wherein the nucleic acid sequence of the TEV protease mutant is shown as SEQ.ID.2, and the amino acid sequence of the TEV protease mutant is shown as SEQ.ID.4. The invention is to integrate the gene of TEV mutant into pET28b vector, transform into BL21 competent cell to express, purify protein by IMAC (immobilized metal chromatography) and SEC (size exclusion chromatography). The TEV mutant shows higher expression, is improved by 6 times compared with the wild-type TEV, and simultaneously improves the heat stability and activity, and after rapid heating at 95 ℃, part of protease is remained and activity is still maintained.

Inventors

• LIU SHIXUAN
• FU BINBIN

Assignees

• 中国药科大学

Dates

Publication Date

20260512

Application Date

20260123

Claims (3)

1. 1. A TEV protease mutant is characterized in that the nucleic acid sequence is shown in SEQ.ID.2, and the amino acid sequence is shown in SEQ.ID.4.
2. 2. Use of a TEV protease mutant according to claim 1 in excision of recombinant protein tags.
3. 3. Use of a TEV protease mutant according to claim 1 for the preparation of a reagent for detection of protein interactions.

Description

TEV protease variant and application thereof Technical Field The invention relates to TEV protease, in particular to a TEV protease variant and application thereof. Background TEV protease is one of the most widely used tool proteases at present. The method is derived from tobacco etching virus, has the molecular weight of 27 kDa and extremely strong sequence specificity, can accurately identify a substrate sequence (ENLYFQG) consisting of seven amino acid residues, and is widely applied to the research fields of excision of recombinant protein tags, detection of protein interaction and the like. Although researchers have also developed various mutants to improve the expression level and enzyme activity, for example, the S219V mutant can not only completely eliminate the self-cleavage phenomenon, but also have greatly improved stability compared with the wild type. However, the mutant is still prone to precipitate at high concentrations, indicating that stability needs to be further improved. The carboxyl terminal of the variant is easy to self-degrade (Parks, 1995), and the heterologous expression level is low. This limits the large-scale application and popularization of the protease. Disclosure of Invention The invention aims to provide a recombinant modified TEV protease mutant which has higher thermal stability, activity and expression quantity. The invention provides a TEV protease mutant, the nucleic acid sequence of which is shown as SEQ.ID.2, and the amino acid sequence of which is shown as SEQ.ID.4. The application of the TEV protease mutant in preparing a excision tool enzyme of a recombinant protein tag. The application of the TEV protease mutant in preparing a detection reagent for protein interaction. The method comprises the following steps: All modifications of the invention are based on Super TEV, as shown in SEQ.ID.1, we see as wild type; The invention has truncations of amino acids 1-10 at the N-terminal compared with the wild-type TEV protease and has the following mutations, The 12 th amino acid is mutated from asparagine (N) to aspartic acid (D), The 52 th amino acid is mutated from asparagine (N) to aspartic acid (D), The 73 rd amino acid is mutated from glutamine (Q) to glutamic acid (E), The 104 th amino acid is mutated from glutamine (Q) to glutamic acid (E), The 105 th amino acid is mutated from arginine (R) to lysine (K), Mutation of asparagine (N) to aspartic acid (D) at position 115, The 117 th amino acid is mutated from glutamine (Q) to glutamic acid (E), Mutation from glutamine (Q) to glutamic acid (E) at position 145, Mutation of 185 th amino acid from asparagine (N) to aspartic acid (D), Mutation of amino acid 193 from glutamine (Q) to glutamic acid (E), The 197 th amino acid is mutated from glutamine (Q) to glutamic acid (E), The 203 th position is mutated from arginine (R) to lysine (K). The specific controls are as follows: 。 table 1 TEV nucleotide sequence of protease: 。 table 2 shows amino acid sequence table of TEV protease: 。 Note that HHHHHH represents a 6 XHis-tag. The invention has the following key points: 1) TEV protease truncation and mutation design. 2) Higher expression level, thermostability and enzyme activity. 3) After rapid heating at 95 ℃, activity is still maintained. The invention has the beneficial effects that the gene of the TEV mutant is integrated into a pET28b vector and transformed into BL21 competent cells for expression, and the protein is purified by IMAC (immobilized metal chromatography) and SEC (size exclusion chromatography). TEV mutants show a 6-fold increase in expression compared to wild-type TEV (here wild-type refers to the more active SuperTEV variant) and also an increase in their thermostability (about 10 ℃ C.) and activity. The data indicate that under rapid heating conditions at 95 ℃, a portion of the protease remains and remains active. Drawings FIG. 1 is a plasmid map of wild-type TEV (A) and mutant TEV (B); FIG. 2 is a molecular sieve chromatogram of wild-type TEV (A) and mutant TEV (B); FIG. 3 is a graph of purified protein glues of wild-type TEV (A) and mutant TEV (B); FIG. 4 is a thermal stability gel diagram of wild-type TEV (A) and mutant TEV (B); FIG. 5 shows the comparison of the activities of wild-type TEV (A) and mutant TEV (B) at 1-24h, wherein the left panel shows the results of wild-type TEV after 1, 2, 4, 8, 24 hours cleavage and the right panel shows the results of mutant after 1, 2, 4, 8, 24 hours cleavage; FIG. 6 is a Mies plot of wild-type TEV and mutant TEV; FIG. 7 shows cleavage activity of wild-type TEV and mutant at 37, 45, 55 ℃, A is a protein gel plot, B is a fluorescent plot; FIG. 8 is a comparison of two hours of cleavage of wild-type and mutant TEV at 4 ℃ (with DTT), 30 ℃ (with DTT) and 30 ℃ (without DTT); FIG. 9 is a comparison of cleavage activity of wild-type and mutant TEV after heating at 30 ℃, 45 ℃, 55 ℃ and 95 ℃. Detailed Description Example 1 construction of plasmid: The method comprises the