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Method for screening protease variant, and protease variant obtained by method

A protease variant and protease technology, applied in the protein field, can solve problems such as TEV protease screening methods that have not been reported yet

Active Publication Date: 2020-04-17
SHANGRAO CONCORD PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Not yet reported the screening method of TEV protease as the present application

Method used

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  • Method for screening protease variant, and protease variant obtained by method
  • Method for screening protease variant, and protease variant obtained by method
  • Method for screening protease variant, and protease variant obtained by method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Embodiment 1: Obtaining of TEV protease variant

[0108] 1. Construction of large-capacity TEV protease random mutation library

[0109] 1.1 Experimental materials:

[0110] Escherichia coli TG1: supE hsdΔ5thiΔ (lac-proAB)F’[traD36proAB+lacIq lacZΔM15], purchased from Beijing Baoke Shiwei’an Company. Phagemid vector pHEN1 (purchased from BioVector NTCC Plasmid Vector Strain Cell Gene Collection Center, Cat. No. Biovector786623). DNA polymerase, T4 DNA ligase, and restriction endonuclease were purchased from Yingwei Jieji Trading Co., Ltd. Plasmid extraction kit and agarose gel DNA recovery kit were purchased from Tiangen (Beijing) Biotechnology Co., Ltd. Random Mutagenesis Kit (GeneMorph II Random Mutagenesis Kit) was purchased from Agilent Technologies. Primer synthesis and gene sequencing were completed in Nanjing GenScript Biotechnology Co., Ltd.

[0111] 1.2. TEVP random mutation library construction

[0112] 1.2.1 Preparation of TEVP DNA fragments by random m...

Embodiment 2

[0176] Embodiment 2: Preparation of ACTH polypeptide with TEVP-ACTH fusion protein

[0177] 1. Construction of TEVP-ACTH fusion protein expression vector

[0178] In Example 1, the carboxyl terminus of ACTH released by fusion proteolysis has a His tag, which is required to be removed in actual production. Therefore, a stop codon needs to be introduced downstream of the ACTH gene by PCR. The specific process is: using the plasmid with TEV protease variant 12D with the best effect in step 3 of Example 1 as a template, the Avi-TEVP-sTEV-ACTH region in the open reading frame of the vector is amplified together by PCR (TEVP variant 12D sequence SEQ ID NO.5, ACTH gene sequence SEQ ID NO.13). Amplify with KOD-plus DNA high-fidelity polymerase (Toyobo), the amplification program is: 95°C pre-denaturation for 2min, 98°C denaturation for 10s, 60°C annealing for 30s, 68°C extension for 1min 12s, 30 cycles of amplification) , a stop codon is introduced downstream of the gene. Primers ...

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Abstract

The present invention relates to a method for screening a protease variant, and the protease variant obtained by the method, wherein the protease variant has specific properties.

Description

[0001] This application claims the priority rights of the Chinese invention patent application No. 201811177105.9 submitted on October 10, 2018, with the invention title "TEV protease variant, its fusion protein, its preparation method and use". technical field [0002] The invention relates to the field of proteins, in particular to a method for screening TEV protease variants and the TEV protease variants. Background technique [0003] TEV protease (TEV Protease) is a 27kDa active domain derived from the Nla protease of tobacco etch virus (TEV), and its amino acid sequence is shown in SEQ ID NO.1. TEV protease has strong site specificity and can recognize EXXYXQ (G / S) seven amino acid sequence, the most commonly used sequence is Glu-Asn-Leu-Tyr-Phe-Gln-Gly (or ENLYFQG), its cleavage site Between glutamine Gln (P1) and glycine Gly (P1') (that is, between P1 and P1'), its sequence specificity is much higher than that of proteases such as thrombin, factor Xa, and enterokinase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50C07K19/00C12N15/57C12N15/62C12N15/70
CPCC12N9/506C12N9/6424C07K14/695C12N15/70C12N15/62C12Y304/21009C07K2319/50C07K2319/21C12Y304/22044C12N9/50
Inventor 刘日魁邹晓龙万江华
Owner SHANGRAO CONCORD PHARMA CO LTD
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