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Method for producing recombinant TEV protease in escherichia coli

A technology of Escherichia coli and a production method, which is applied to the production field of recombinant TEV protease in Escherichia coli, can solve the problems of biological activity, low purity, complicated steps and high cost, and achieves the effects of saving experimental steps, improving purity and reducing cost.

Inactive Publication Date: 2018-11-09
通用生物(安徽)股份有限公司
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Problems solved by technology

[0005] At present, TEV protease on the market has generally low biological activity and purity, and the common production method of TEV protease has cumbersome steps and high cost

Method used

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Examples

Experimental program
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Embodiment 1

[0031] The production method of recombinant TEV protease in escherichia coli comprises the following steps:

[0032] S1. The construction of PET-28a-TEV protease expression plasmid is divided into the following steps:

[0033] a. According to the codon-optimized gene sequence, the TEV protease directly performs gene synthesis;

[0034] b. The PET-28a carrier plasmid is linearized by enzyme digestion, and the linearized product and the gene synthesis product are seamlessly constructed to form a recombinant product;

[0035] c. Take 20ul of the recombinant product and transfer it to TOP10 competent cells, let it stand on ice for 30min, then heat shock at 42°C for 90s, let stand on ice for 2min, then add 600ul of LB liquid medium, shake it at 37°C and 200rpm Incubate for 45 minutes, spread on LB plates containing 50ug / mL kanamycin, and finally place in a 37°C incubator for overnight cultivation;

[0036] d. Use a sterilized toothpick to randomly pick four colonies from the LB p...

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Abstract

The invention discloses a method for producing recombinant TEVprotease in escherichia coli. The method comprises the following steps: S1) construction of PET28aTEV protease expression plasmid; S2) inducing expression and identification of TEV protease; and S3) purification and identification of TEV protease. The invention adopts the escherichia coli expression system to realize the expression of recombinant TEV protease in escherichia coli by designing an expression vector and then adopts affinity chromatography and Superdex 75 gel filtration chromatography to purify the target protease, thusgreatly shortening the purification process and time of the target protease, improving the purity, yield and activity of the protease, saving experimental steps and reducing cost. The method providedby the invention lays a foundation for the development and production of other protein tool enzymes.

Description

technical field [0001] The invention relates to the field of polymer reaction, in particular to the production method of recombinant TEV protease in Escherichia coli. technical background [0002] Currently, low protein yield and activity are technical bottlenecks for heterologous protein expression, especially toxic protein expression. With the development of biotechnology, researchers have applied fusion expression strategies to overcome the difficulties in the expression of toxic and insoluble proteins. [0003] TEV protease is derived from the recombinant protease of tobacco etch virus (TEV) N1a, and this protease is used to excise the affinity tag of fusion protein after purification. TEV protease has strong site specificity and can recognize the seven amino acid sequence (Glu-Asn-Leu-Tyr-Phe-Gln-Gly) of EXXYXQ (G / S), the most common one is ENLYFQG, and its cleavage site is at between glutamine and glycine or serine. The enzyme is active in a wide range of pH 5.5-8.5...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N9/50
CPCC12N9/503C12N15/70
Inventor 雍金贵杜攀李森
Owner 通用生物(安徽)股份有限公司
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