Gene knockout Escherichia coli beneficial to recombinant protein exocytosis and application thereof

A technology of Escherichia coli and gene knockout, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of increasing downstream costs and low extracellular expression, and achieve the effects of low cost, enhanced extracellular secretion, and high efficiency

Inactive Publication Date: 2015-08-19
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The fructosidase is expressed in the Escherichia coli system, and the intracellular expression is relatively large, which i...

Method used

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  • Gene knockout Escherichia coli beneficial to recombinant protein exocytosis and application thereof
  • Gene knockout Escherichia coli beneficial to recombinant protein exocytosis and application thereof
  • Gene knockout Escherichia coli beneficial to recombinant protein exocytosis and application thereof

Examples

Experimental program
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Effect test

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 penicillin G acylase derived from Escherichia coli was cloned into the plasmid pET22b(+) to obtain 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 recombinant protein exocytosis and application thereof, relating to the field of modification of Escherichia coli genetic group genes. The gene knockout Escherichia coli beneficial to recombinant protein exocytosis is prepared by knocking out lipopolysaccharide synthesis related genes in Escherichia coli. The invention also provides a construction method of the gene knockout Escherichia coli and application of the gene knockout Escherichia coli in preparing extracellular soluble proteins. The preparation method is simple, and has the advantages of high efficiency and low cost. After the lipopolysaccharide-synthesized related genes are knocked out, the permeability of the pericellular membrane is enhanced, thereby obviously enhancing the recombinant protein exocytosis.

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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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 UNIV OF TECH
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