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Construction method for double-gene mutation escherichia coli used for secretory expression of recombinant protein

A technology for recombining Escherichia coli and Escherichia coli, which is applied in the field of transforming Escherichia coli genetic genome, to achieve the effect of canceling the cell wall breaking process, simplifying the process and reducing pyrogen pollution

Inactive Publication Date: 2012-12-19
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

e.g. Escherichia coli lpp The mutant can leak the periplasmic space protein into the culture medium without significantly affecting the normal growth of the strain, but there is still a lot of room for improvement in the leakage ratio and production of the target protein

Method used

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  • Construction method for double-gene mutation escherichia coli used for secretory expression of recombinant protein

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Effect test

Embodiment 1

[0050] A method for constructing Escherichia coli for secretory expression of recombinant proteins, comprising the following steps:

[0051] A. pal with wxya The construction of the secreted bacterial strain of double gene mutation comprises the following steps:

[0052] a. Build pal Genetically mutated secretory strains:

[0053] (1) Use a pre-designed pal Gene knockout primers palH+PF and palH+PR, using the resistance marker gene plasmid pKD3 as a template, carry out polymerase chain reaction to synthesize a specific PCR product to obtain pal Gene targeting fragment, both ends of the targeting fragment are 50bp pal The homologous sequence of the gene, the middle is the chloramphenicol resistance marker sequence, and the obtained PCR product is obtained using a PCR product purification kit to obtain a purified high-concentration targeting fragment of 20 ng / μl;

[0054] (2) Use 10mmol / L L-arabinose to induce the starting Escherichia coli strain JM109(DE3) containing ...

Embodiment 2

[0063] A method for constructing Escherichia coli for secretory expression of recombinant proteins, comprising the following steps:

[0064] A. wxya with lpp The construction of the secreted bacterial strain of double gene mutation comprises the following steps:

[0065] a. Build lpp Genetically mutated secretory strains:

[0066] (1) Use a pre-designed lpp Gene knockout primers lppH+PF and lppH+PR, using the resistance marker gene plasmid pKD3 as a template, carry out polymerase chain reaction to synthesize specific PCR products to obtain lpp Gene targeting fragment, both ends of the targeting fragment are 50bp lpp The homologous sequence of the gene, the middle is the chloramphenicol resistance marker sequence, and the obtained PCR product is obtained using a PCR product purification kit to obtain a purified high-concentration targeting fragment of 20 ng / μl;

[0067] (2) Use 10mmol / L L-arabinose to induce the starting Escherichia coli strain JM109(DE3) containing the ...

Embodiment 3

[0076] A method for constructing Escherichia coli for secretory expression of recombinant proteins, comprising the following steps:

[0077] A. pal with wxya The construction of the secreted bacterial strain of double gene mutation comprises the following steps:

[0078] a. Build pal Genetically mutated secretory strains:

[0079] (1) Use a pre-designed pal Gene knockout primers palH+PF and palH+PR, using the resistance marker gene plasmid pKD3 as a template, carry out polymerase chain reaction to synthesize a specific PCR product to obtain palGene targeting fragment, both ends of the targeting fragment are 50bp pal The homologous sequence of the gene, the middle is the chloramphenicol resistance marker sequence, and the obtained PCR product is obtained using a PCR product purification kit to obtain a purified high-concentration targeting fragment of 20 ng / μl;

[0080] (2) Use 10mmol / L L-arabinose to induce the starting Escherichia coli strain JM109(DE3) containing...

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Abstract

The invention discloses a construction method for double-gene mutation escherichia coli used for secretory expression of recombinant protein. According to the invention, pre-designed primers for the double-knockout-genes of cell membrane protein and cell wall synthetase gene are used for a PCR reaction so as to obtain gene targeting fragments of the double-knockout-gene, wherein two ends of each gene targeting fragment are 10 to 250 bp homologous sequences of a specific target gene and the middle part of each gene targeting fragment is a resistance labeled sequence; the fragments are respectively integrated into chromosomes of the host escherichia coli through electrotransformation so as to obtain a secretory double-gene mutation escherichia coli strain. The method provided by the invention increases the expression of recombinant exogenous proteins, reduces intracellular degradation of target proteins, decreases intracellular accumulation of the recombinant exogenous proteins and eradicates formation of inclusion bodies; the method discards a breaking process for cell walls, reduces pyrogen and simplifies separation and purification; thus, the purposes of reducing production cost and improving protein expression and product quality are achieved.

Description

technical field [0001] The invention relates to the field of genetically modified Escherichia coli, in particular to a method for constructing a double-gene mutant Escherichia coli used for secretory expression of recombinant proteins. Background technique [0002] In recent years, the recombinant protein drug market has developed rapidly and has become an important high-tech industry. The important recombinant protein drugs in the market mainly include: human parathyroid hormone, B lymphocyte stimulating factor, human epidermal growth factor, γ-interferon, hemolysin and various antibodies. Escherichia coli remains the most widely used host system for the production of various recombinant proteins. In the E. coli host system, according to the spatial location of the recombinant protein after synthesis, it can be divided into intracellular expression and secretory expression. Recombinant proteins expressed intracellularly in E. coli often form inclusion bodies, which are in...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12R1/19
Inventor 童望宇陈昭元谢丽
Owner ANHUI UNIVERSITY
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