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TEV protease variant, fusion protein, preparation method and uses thereof

A technology of protease variants and fusion proteins, which is applied in the fields of fusion with protease sites, botanical equipment and methods, biochemical equipment and methods, etc., which can solve the problems of unfavorable cost control, many process steps, and low efficiency

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

The disadvantage of this production method is that there are too many process links, the efficiency is not high, and it is not conducive to cost control

Method used

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  • TEV protease variant, fusion protein, preparation method and uses thereof
  • TEV protease variant, fusion protein, preparation method and uses thereof
  • TEV protease variant, fusion protein, preparation method and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Embodiment 1: Obtaining of TEV protease variant

[0107] 1. Construction of large-capacity TEV protease random mutation library

[0108] 1.1 Experimental materials:

[0109] Escherichia coli TG1: supE hsdΔ5thiΔ(lac-proAB)F’[traD36proAB+lacIqlacZΔM15] was purchased from Beijing Baoke Shiwei’an Company. Phagemid vector pHEN1 (purchased from BioVectorNTCC 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.

[0110] 1.2. TEVP random mutation library construction

[0111] 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 invention provides a TEV protease variant, a fusion protein, a preparation method and uses thereof, particularly a screened TEV protease variant with unique properties, and a fusion protein. According to the invention, polypeptides can be rapidly and efficiently prepared through TEV protease variant and polypeptide fusion expression so as to solve the problems existing in the current polypeptide recombinant production process.

Description

[0001] This application claims the priority right of Chinese Invention Patent Application No. 201811177281.2 filed on October 10, 2018. technical field [0002] The present invention relates to the protein field, in particular to TEV protease. 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. [0004] TEV protease can tolerate a wide range of pH (pH 4-8.5) and temperature (4-34 ℃), and some common additives that increase protein solubility or stability (e...

Claims

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Application Information

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