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A method for expressing hmgb1 A-box protein using SUMO system

An a-box and sumo-a-box technology, applied in the field of protein engineering, can solve the problems of unsatisfactory HMGB1A-box yield, unreported HMGB1A-box protein, expensive protease cost, etc., to improve soluble expression level, ensure biological activity, and promote the effect of correct folding

Inactive Publication Date: 2016-09-21
XIN HUA HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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Problems solved by technology

Although the solubility and yield of the protein have been improved, the time-consuming, expensive protease costs and the risk of protease hydrolysis of the target protein often fail to obtain a satisfactory yield of HMGB1 A-box
[0005] However, there is no report on the technology of efficient and soluble expression of HMGB1 A-box protein using the SUMO system

Method used

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  • A method for expressing hmgb1 A-box protein using SUMO system
  • A method for expressing hmgb1 A-box protein using SUMO system
  • A method for expressing hmgb1 A-box protein using SUMO system

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Embodiment 1

[0024] 1. Experimental design

[0025] Using the plasmid containing HMGB1 as a template, design primers to amplify the A-Box domain, subclone the amplified target gene A-Box into the pSumo-Mut expression vector between Stu I and Hind III, and fuse the N-terminal of the target gene The Sumo tagged protein was obtained, and the pSumo-Mut-A-Box plasmid was obtained. Transformation of ArcticExpress with recombinant plasmids TM (DE3) Prokaryotic host bacteria, using IPTG to induce the expression of the target protein. Optimize the expression conditions, adjust the induction conditions to 11 degrees, and detect that the sumo-A-Box fusion protein is distributed in the supernatant, and obtain the sumo-A-Box fusion protein through Ni affinity purification, and then enzymatically digest sumo protease to fuse sumo The protein is separated from A-Box, and then subjected to Ni column affinity purification to remove residual Sumo-A-Box fusion protein, Sumo fusion tag and sumo protease, a...

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Abstract

The present invention relates to a method for expressing the HMGB1 A-box protein using the SUMO system, comprising the following steps: using a plasmid containing HMGB1 as a template, designing primers to amplify the A-Box domain, substituting the amplified target gene A-Box Cloned into the pSumo-Mut expression vector between Stu I and Hind III to obtain the pSumo-Mut-A-Box plasmid; use the recombinant plasmid to transform ArcticExpress TM (DE3) Prokaryotic host bacteria, using IPTG to induce expression of the target protein; sumo protease enzymatic hydrolysis to obtain high-purity A-Box protein. The invention has the advantages that: the HMGB1 A-box fusion protein is efficiently, stably and solublely expressed by using the SUMO expression system, and a mature protein with high purity can be obtained after cleavage by SUMO protease I, and no amino acid residue remains.

Description

technical field [0001] The invention relates to the field of protein engineering, in particular, it is a technique for efficiently and solublely expressing HMGB1 A-box protein by using a SUMO system. Background technique [0002] The production of HMGB1 A-box currently mainly utilizes protein engineering technology and fusion technology. [0003] 1. Using protein engineering technology, use Escherichia coli as a preferred host cell for industrial protein production by recombinant technology to produce HMGB1 A-box. Although the Escherichia coli expression system has the advantages of high expression, easy cultivation and operation, and low production cost, it is still difficult to directly obtain a large amount of soluble HMGB1 A-box by using this expression system. Predisposed to form "inclusion bodies," insoluble aggregates that are biologically inactive. In addition, even if a large number of inclusion bodies are obtained, in order to obtain biologically active proteins,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/12C07K14/47
Inventor 葛文松陈颖伟范建高
Owner XIN HUA HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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