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Expression vector, expression system and application of recombinant TEV protease

A technology for expressing vectors and recombinant proteins, which is applied in the biological field and can solve problems such as low protein stability

Pending Publication Date: 2020-10-20
GUIZHOU INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since the serine at position 219 is not mutated, the protein is prone to self-degradation, and the protein has a problem of low stability.

Method used

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  • Expression vector, expression system and application of recombinant TEV protease
  • Expression vector, expression system and application of recombinant TEV protease
  • Expression vector, expression system and application of recombinant TEV protease

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Embodiment 1

[0033] 1. Experimental materials, reagents and experimental instruments

[0034] 1.1 Experimental materials

[0035] E.coli BL21 (DE3) is a strain preserved in our laboratory.

[0036] The recombinant plasmid pET-21a-TEVp was donated by Dr. Michel Vincent, University of Laval, Canada. The schematic diagram of the structure of plasmid pET-21a is attached figure 1 shown.

[0037] The four-point mutant TEVp (T17S, L56V, S135G and S219V) gene was inserted into the multiple cloning site of the pET-21a plasmid, and the recombinant plasmid pET-21a-TEVp was transformed into E.coli BL21 (DE3) and screened positive by Amp resistance Cloning, IPTG induced expression to produce His-TEVp recombinant protein.

[0038] 1.2 Main reagents and sources

[0039] Ni 2+ -NTA-Sepharose is a product of Amersham Company. IPTG reagent is the product of Sigma Company. Yeast extract and peptone were from Oxiod. The plasmid extraction kit was from Tiangen Company. All other reagents obtained fr...

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Abstract

The invention relates to the field of biotechnology, and in particular to an expression vector, expression system and application of a recombinant TEV protease. The recombinant TEV protease expressionvector is obtained by inserting a His-TEVp recombinant protein into a pET-21a vector. The recombinant TEV protease expression system comprises introducing the recombinant TEV protease expression vector into E. coli to obtain a recombinant TEV protease expression strain. The His-TEVp recombinant protein is generated by inserting a four-point mutant TEVp gene into the pET-21a plasmid to obtain a recombinant plasmid pET-21a-TEVp. The TEV protease expression system is utilized to express the TEV protease, and then a large amount of high-purity TEV protease can be obtained by extraction and purification; the purity of the TEV protease can reach more than 90%; and the concentration of TEVp in the production bacterial liquid can reach 1.42 mg / ml, and high-purity TEV protease of more than 40 mg can be obtained in each liter of bacterial liquid. The expression vector and expression system of the recombinant TEV protease provided by the present invention lay a foundation for the popularizationand application of the TEV protease.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant TEV protease expression vector, expression system and application. Background technique [0002] Tobacco etch virus protease (Tobacco Etch Virus protease, TEVp) is a kind of recombinant protease derived from tobacco etch virus (TEV) Nla, which is widely used in the field of biochemistry because of its remarkable specificity. The standard TEVp is a C-type cysteine ​​protease with 242 amino acids and a molecular weight of 27KDa, which can effectively remove the tag of the fusion protein from the C-terminus and N-terminus of the recombinant protein. Compared with other proteases, TEVp has obvious advantages. First, TEVp is highly specific and can effectively recognize the seven amino acid sequence EXXYXQ(G / S), among which ENLYFQG is the most common specific amino acid sequence recognition sequence, and the cleavage site is between glutamine and glycine or serine; Secondl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N9/50C12N1/20C12R1/19
CPCC12N15/70C12N9/50
Inventor 朱国飞马超刘丽萍余凯周安
Owner GUIZHOU INST OF TECH
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