Escherichia coli hydrotropic expression plasmid and construction method and application thereof

A technology of Escherichia coli and expression plasmid, which is applied in the field of bioengineering to achieve the effects of high-efficiency soluble expression and good solubilization effect

Pending Publication Date: 2021-11-09
GUANGXI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods in the prior art still need to be further improved in improving the expression of foreign proteins

Method used

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  • Escherichia coli hydrotropic expression plasmid and construction method and application thereof
  • Escherichia coli hydrotropic expression plasmid and construction method and application thereof
  • Escherichia coli hydrotropic expression plasmid and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] To coelicolor DNA as a template to amplify groEL1, groES1 and groEL2 partner genes. Amplification reaction system is: template DNA 1μL, upstream and downstream of each primer 1μL, 2 × Phanta Max MasterMix 25μL, ddH 2 O 22μL. Amplification conditions were: 95 ℃ denaturation 5min; 95 ℃ 30s, 60 ℃ 30s, 72 ℃ 2min, 35 cycles; 72 ℃ 10min. GroEL1 amplified gene upstream primer as shown in SEQ ID NO.4; groEL1 amplified gene downstream primer shown as SEQ ID NO.5. As shown in SEQ ID NO.6 groES1 amplified gene upstream primer; groES1 downstream primer amplified gene was shown as SEQ IDNO.7. GroEL2 amplified gene upstream primer as shown in SEQ ID NO.8; groEL2 amplified gene downstream primer as shown in SEQ ID NO.9.

[0043] After the end of the PCR amplification reaction PCR product subjected to 1% agarose electrophoresis. The results, pGro7 3500bp in a specific band; groEL1 1600bp in a specific band; groES1 300bp in a specific band; groEL2 1600bp in a specific band.

[0044] Compani...

Embodiment 2

[0050] The plasmid vector pET28a-dnmQ chemical conversion to pGroE / BL21 (DE3) competent cells, 37 [deg.] C obtained in Production Example 1, culture incubator 12h, to give BL21 (DE3) / pGroE / pET28a-dnmQ. The ratio of BL21 (DE3) / pGroE / pET28a-dnmQ 1:50 dilution by volume inoculated in TB medium, 37 [deg.] C with shaking until an OD600 of 0.7, a molar concentration of added 0.1M isopropyl -β-D- thiogalactopyranoside and concentration of 0.5g / ml of L- arabinose, induce the expression of 37 [deg.] C 4h, cells were collected expression. Expression of bacterial cells by sonication, centrifuged at 12000 rpm 30min lysing supernatant was collected, to give expression product. Soluble expression of expression products by SDS-PAGE detection of glycosyltransferase dnmQ. Such as Figure 4 Indicated.

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Abstract

The invention relates to the technical field of bioengineering, and particularly relates to an escherichia coli hydrotropic expression plasmid and a construction method and application thereof. According to the invention, the construction method of the hydrotropic expression plasmid comprises the following steps: (1) by taking streptomyces DNA as a template, amplifying to obtain partner genes groEL1, groES1 and groEL2; and (2) cloning the partner genes groEL1, groES1 and groEL2 to an escherichia coli plasmid vector pGro7 of which groEL and groES gene segments are deleted so as to obtain the hydrotropic expression plasmid pGroE. The pGroE plasmid disclosed by the invention and a dnmS and / or dnmQ gene coded glycosylated transferase are co-expressed; compared with the solubility of a pGro7 plasmid, the solubility of the pGroE plasmid can be remarkably improved; and the development of the hydrotropic expression plasmid provides a good solution for solving the inclusion body problem during gene expression.

Description

Technical field [0001] The present invention relates to the field of biological engineering technology, particularly to a solubilizing the E. coli expression plasmid and its construction and method of application. Background technique [0002] In vitro recombinant DNA techniques such that expression of the protein as possible. E. its easy genetic manipulation, propagation speed, high expression of recombinant protein production, culture and low cost, other than the heterologous protein expression host, a powerful tool in molecular biology research. However, E. coli transcriptional modified proteins can not be achieved after intracytoplasmic reducing environment is not conducive to disulfide bond formation and stability, resulting in part of the recombinant protein can not be correctly folded, and thus easy to form biologically inactive insoluble inclusion bodies. [0003] Means for Solving the Problems have reduced expression of the inclusion body temperature, or to replace a wea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/64C12N15/31C12N15/54C12N9/10
CPCC12N15/70C07K14/36C12N9/1048
Inventor 潘丽霞周会敏杨登峰阳丽艳李红亮
Owner GUANGXI ACAD OF SCI
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