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A gene-knockout Escherichia coli beneficial to extracellular secretion of recombinant protein and its application

A technology of Escherichia coli and gene knockout, which is applied in application, genetic engineering, plant gene improvement, etc., can solve the problems of low extracellular expression and increase downstream costs, and achieve enhanced extracellular secretion, low cost, and improved yield and purity Effect

Inactive Publication Date: 2017-12-22
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fructosidase is expressed in the Escherichia coli system, and the intracellular expression is relatively large, which is easy to form inclusion bodies, and the extracellular expression is very small, which significantly increases the downstream cost

Method used

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  • A gene-knockout Escherichia coli beneficial to extracellular secretion of recombinant protein and its application
  • A gene-knockout Escherichia coli beneficial to extracellular secretion of recombinant protein and its application
  • A gene-knockout Escherichia coli beneficial to extracellular secretion of recombinant protein and its application

Examples

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Embodiment 1

[0038] This embodiment illustrates the construction process of Escherichia coli that knocks out the waaF gene, which specifically includes the following steps:

[0039] (1) The gene waaF (SEQ ID NO: 1) of lipopolysaccharide heptose transferase II in Escherichia coli BL21 (DE3) was first cloned by PCR using waaF gene cloning primers waaF-F and waaF-R, and connected to the pMD18T vector A recombinant plasmid was constructed on the above and named pMD18T-waaF;

[0040] (2) Using the pMD18T-waaF recombinant plasmid obtained in step (1) as a template, perform reverse PCR with reverse primers fxwaaF-F and fxwaaF-R, remove the sequence in the middle of waaF, and amplify the two ends of the waaF gene with a length of about 220bp The homologous sequence of the inverse PCR product and the resistance fragment dif-Gm r -dif link, i.e. in the resistant fragment dif-Gm r The two ends of -dif are respectively connected with the homologous sequence of the waaF gene to obtain the fusion gene...

Embodiment 2

[0046] This example illustrates the method for constructing fructosidase-producing bacteria using Escherichia coli knockout of the waaF gene.

[0047] The gene fragment Fru of fructosidase with its own signal peptide (disclosed in the invention patent of patent No. ZL201210185733.8, named an organic solvent-resistant glycosidase Fru6 and its gene and application, the sequence is as SEQ in this patent ID NO: shown in 1) was cloned into the plasmid pET22b (+) to obtain the recombinant plasmid pET22b (+)-Fru, and then transformed into the Escherichia coli strain (prepared in Example 1) that knocked out the waaF gene to obtain Escherichia coli BL21 ( DE3)::ΔwaaF / pET22b(+)-Fru.

Embodiment 3

[0049] This example illustrates the method for constructing a penicillin G acylase-producing bacterium using Escherichia coli in which the waaF gene has been knocked out.

[0050] The gene fragment pga (Cheng TF et al. Protein Expression and Purification, 2006,46 (1): 107-113.) of the penicillin G acylase derived from Escherichia coli was cloned in the plasmid pET22b (+), and the recombinant plasmid pET22b ( +)-pga, and then transformed into the Escherichia coli strain (prepared in Example 1) where the waaF gene was knocked out to obtain Escherichia coli BL21(DE3)::ΔwaaF / pET22b(+)-pga.

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Abstract

The invention provides a gene-knockout Escherichia coli beneficial to the extracellular secretion of recombinant proteins and its application, and relates to the field of genetic genome transformation of Escherichia coli. The gene-knockout Escherichia coli which is beneficial to the extracellular secretion of the recombinant protein is obtained by knocking out the genes related to lipopolysaccharide synthesis in the Escherichia coli. The present invention also provides a construction method of the gene-knockout Escherichia coli and an application of the gene-knockout Escherichia coli in preparing extracellular soluble proteins. The preparation method of the gene-knockout Escherichia coli of the present invention is simple, high in efficiency and low in cost, and the permeability of the outer membrane of the cell is increased after the gene knockout of lipopolysaccharide synthesis is knocked out, thereby significantly enhancing the extracellular secretion of the recombinant protein.

Description

technical field [0001] The invention relates to the field of genetically modified Escherichia coli, in particular to a gene-knockout Escherichia coli beneficial to the extracellular secretion of recombinant proteins and its application. Background technique [0002] The E. coli system is suitable for the production of proteins that do not require post-translational modification. Since Escherichia coli lacks the cofactors required in the protein folding process, the intermediates tend to accumulate in large quantities to form inclusion body precipitates. In order to obtain active proteins, the inclusion bodies must undergo tedious processes such as renaturation. The fructosidase is expressed in the Escherichia coli system. The intracellular expression is large and inclusion bodies are easy to form, and the extracellular expression is very small, which significantly increases the downstream cost. [0003] Lipopolysaccharide (LPS) is a unique structural and functional molecule...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/54C12N9/26C12N9/84C12R1/19
Inventor 何冰芳朱芸储建林周有治吴斌柏中中钦松姜天玥
Owner NANJING TECH UNIV
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