TEV protease mutant, gene, biomaterial, preparation method, reagent, or reagent kit and application

A biomaterial, mutant technology, applied in microorganism-based methods, biochemical equipment and methods, botanical equipment and methods, etc., can solve the problems of low yield and water solubility, reduced protease activity and stability, etc. Activity and stability, improved water solubility, good stability and specificity

Active Publication Date: 2018-12-21
生工生物工程(上海)股份有限公司
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Problems solved by technology

[0005] There are certain defects in the expression and purification of wild-type TEV in Escherichia coli. When the wild-type TEV is expressed, it will undergo intramolecular self-cleavage at its own specific site, thereby greatly reducing the activity and stability of the protease, and When expressed in E. coli, most of them exist in the form of inclusion bodies, and the yield and water solubility are very low

Method used

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  • TEV protease mutant, gene, biomaterial, preparation method, reagent, or reagent kit and application
  • TEV protease mutant, gene, biomaterial, preparation method, reagent, or reagent kit and application
  • TEV protease mutant, gene, biomaterial, preparation method, reagent, or reagent kit and application

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preparation example Construction

[0058] The present invention also provides a preparation method of the rTEV, comprising expressing the gene encoding the rTEV in a host.

[0059] The preparation method can be performed by expressing the above-mentioned gene encoding the rTEV in a host, for example, but not limited to, an Escherichia coli expression system, a yeast expression system, an insect expression system, a plant expression system or a mammalian expression system.

[0060] In some preferred embodiments, the gene expressing the rTEV is first inserted into the recombinant plasmid obtained by the pET-28a vector; then the recombinant plasmid is introduced into E. rTEV.

[0061] The present invention also provides a reagent or a kit containing the rTEV, and the reagent or the kit may also include reagents and / or consumables matched with the rTEV, for example, but not limited to, other enzyme-cutting DNA Protease, Buffer for dissolving protease, SDS-PAGE related reagents or experimental consumables for more ...

Embodiment 1

[0066] This embodiment provides a TEV protease mutant named rTEV, and TEVw is the wild-type TEV protease, which has the mutation sites as shown in the following table:

[0067] Table 1 Mutation sites of rTEV

[0068] amino acid position

TEVw amino acid name

rTEV amino acid name

17

T

S

56

L

V

68

N

D

77

I

V

135

S

G

219

S

V

233

Q

H

[0069] The preparation method of rTEV is as follows:

[0070] The whole gene was synthesized according to the nucleotide sequence of rTEV in Table 1, and subcloned into the final expression vector pET-28a with Nde I as the N-terminal restriction site and Xho I as the C-terminal restriction site. After sequencing by Shanghai Sangon Bioengineering Technology Service Co., Ltd., it was finally verified that the obtained recombinant plasmid sequence was consistent with the sequence of the theoretical seven-mutant TEV protease.

[0071] Expression an...

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Abstract

The invention provides a TEV protease mutant, a gene, a biomaterial, a preparation method, a reagent, or a reagent kit and application, and relates to the technical field of genetic engineering. Compared with a protease mutant having an amino acid sequence shown as SEQ ID NO.1, the TEV protease mutant is mutated into at least seven amino acid residues, for example 17th site is mutated into S fromT, the 56th site is mutated into V from L, the 68th site is mutated into D from N, the 77th site is mutated into V from I, the 135th site is mutated into G from S, the 219th site is mutated into V from S, and the 233th site is mutated into H from Q. The TEV protease mutant has favorable enzymatic activity, stability and specificity. The gene, the biomaterial and the preparation method of the TEV protease mutant, or the reagent or the reagent kit containing the TEV protease mutant, the application of the technical scheme, and the TEV protease mutant are all based on the same invention conception.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a TEV protease mutant, gene, biological material, preparation method, reagent or kit and application. Background technique [0002] Prokaryotic expression is widely used in bioengineering. There are countless sources of genes expressed by prokaryotic expression systems, covering research directions in many fields, including protein purification, localization, and functional analysis. According to different expression systems, it can be divided into E. coli expression system, Bacillus subtilis expression system, Streptomyces expression system and so on. Among them, the Escherichia coli expression system is the most mature technology and the most widely used because of its simple culture method, clear expression background, and low expression cost. [0003] However, the Escherichia coli expression system lacks enzymes, cofactors and other substances unique to the post-translatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50C12N15/57C12N15/70C12N1/21C12R1/19
CPCC12N9/506
Inventor 王欢付康初腾
Owner 生工生物工程(上海)股份有限公司
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