Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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

A technology for recombining Escherichia coli and Escherichia coli is applied in the field of genetic modification of Escherichia coli to achieve the effects of simplifying separation and purification, reducing production costs and reducing excessive accumulation in cells

Inactive Publication Date: 2014-06-04
ANHUI UNIVERSITY
View PDF0 Cites 1 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Construction method for double-gene mutation escherichia coli used for secretory expression of recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0051] A. pal and mrcB The construction of the double gene mutant secretory strain includes the following steps:

[0052] a. to build pal 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 specific PCR products, and obtain pal Gene targeting fragment, both ends of the targeting fragment are 50bp pal The gene homologous sequence, the middle is the chloramphenicol resistance marker sequence, and the obtained PCR product is used to obtain a purified high-concentration targeting fragment of 20ng / μl using a PCR product purification kit;

[0054] (2) Use 10 mmol / L L-arabinose to induce the E. coli starting strain JM109(DE3) containing the knockout helper plasmid pKD46 for 2 hours, a...

Embodiment 2

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

[0064] A. mrcB and lpp The construction of the double gene mutant secretory strain includes the following steps:

[0065] a. to build lpp 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, and obtain lpp Gene targeting fragment, both ends of the targeting fragment are 50bp lpp The gene homologous sequence, the middle is the chloramphenicol resistance marker sequence, and the obtained PCR product is used to obtain a purified high-concentration targeting fragment of 20ng / μl using a PCR product purification kit;

[0067] (2) Use 10 mmol / L L-arabinose to induce the E. coli starting strain JM109(DE3) containing the knockout helper plasmid pKD46 for 2 hours, and ...

Embodiment 3

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

[0077] A. pal and mrcB The construction of the double gene mutant secretory strain includes the following steps:

[0078] a. to build pal 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 specific PCR products, and obtain palGene targeting fragment, both ends of the targeting fragment are 50bp pal The gene homologous sequence, the middle is the chloramphenicol resistance marker sequence, and the obtained PCR product is used to obtain a purified high-concentration targeting fragment of 20ng / μl using a PCR product purification kit;

[0080] (2) Use 10 mmol / L L-arabinose to induce the E. coli starting strain JM109(DE3) containing the knockout helper plasmid pKD46 for 2 hours, ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12R1/19
Inventor 童望宇陈昭元谢丽
Owner ANHUI UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com