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Method for accurately and quantitatively regulating and controlling exocytosis level of escherichia coli recombinant protein and application

A technology of Escherichia coli and recombinant Escherichia coli, which is applied in the field of genetic engineering and fermentation engineering, can solve problems such as toxicity, achieve the effects of improving permeability, optimizing application effect, and promoting extracellular secretion

Active Publication Date: 2022-07-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CN105755029A discloses a method based on dacA to improve the extracellular secretion level of Escherichia coli recombinant protein, but D, D-carboxypeptidase itself has the disadvantage of toxicity to the bacteria

Method used

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  • Method for accurately and quantitatively regulating and controlling exocytosis level of escherichia coli recombinant protein and application
  • Method for accurately and quantitatively regulating and controlling exocytosis level of escherichia coli recombinant protein and application
  • Method for accurately and quantitatively regulating and controlling exocytosis level of escherichia coli recombinant protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Knockout of tryptophan synthase gene cluster trpEDCBA in recombinant Escherichia coli

[0068] (1) Using plasmid pKD13 as a template, design primers and PCR amplify the homologous fragment containing the Kan resistance gene for replacing the trpEDCAB sequence, wherein the Kan resistance gene contains FRT sites on both sides to obtain the trpEDCBA sequence integration frame fragment.

[0069](2) Transform the pKD46 plasmid into E.coli BL21(DE3) competent cells to obtain E.coli G0 strain, prepare electrotransformed competent cells, and electro-transform the integration frame fragment obtained in step (1) into E.coli In the G0 competent state, the transformation solution was post-cultured and then coated with LB solid medium containing kanamycin (Kan) and ampicillin to obtain transformant BL21::kan, using primers TrpA-pKD13-FW and TrpE- pKD13-RW was verified by colony PCR amplification to verify whether the trpEDCBA sequence was successfully edited, and the posit...

Embodiment 2

[0073] Example 2 Screening and characterization of stationary phase promoters

[0074] A library of 40 promoters was synthesized (a partial list of promoters is shown in 1) for screening different target promoters with gradient expression levels. Firstly, the lacI gene fragment corresponding to the lac operon on the plasmid pRSFDuet-1 was deleted to obtain the plasmid pRSFDuet-1-ΔlacI. The 40 promoters in the promoter library were used to replace the original T7 promoter in the plasmid pRSFDuet-1-ΔlacI respectively to construct a plasmid library containing 40 different promoters. Using green fluorescent protein (eGFP) as a model protein, different promoters with gradient expression levels were screened. Screening to obtain 5 different eGFP expression intensities (e.g. figure 2 ), and the protein expression level is concentrated in the promoter in the stationary phase (P 25 , P 41 , P 53 , P 69 , P 84 ) for the construction of novel regulatory systems in subsequent stud...

Embodiment 3

[0077] Example 3 Design and application of a new tryptophan operator system

[0078] Design of the new tryptophan operon system: The new tryptophan operon system consists of E. coli cells knocked out of the tryptophan synthase gene cluster trpEDCBA and a recombinant plasmid; the recombinant plasmid contains T7 promoter, SPP-type promoter, Tryptophan synthase gene cluster trpEDCBA, operator region trpO and D,D-carboxypeptidase gene dacA; the operator region trpO has a binding site that can bind to a covalent dimer formed by the repressor protein trpR and tryptophan The T7 promoter regulates the expression of trpEDCBA; the SPP regulates the gene expression of the operator region trpO; the downstream of the operator region trpO is connected to the dacA gene; the transcription directions of the T7 promoter and promoter SPP are opposite; the The nucleotide sequence of the T7 promoter is shown in SEQ ID No. 6; the nucleotide sequence of the SPP type promoter is shown in SEQ ID No. 7...

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Abstract

The invention discloses a method for accurately and quantitatively regulating and controlling the exocytosis level of escherichia coli recombinant protein and application, and belongs to the field of genetic engineering and fermentation engineering. The tryptophan operon constructed in the invention is combined with a thermo-sensitive repression system (TAT) co-expression system to realize stable and normal growth of Escherichia coli in the early growth stage, and the fermentation temperature is controlled and the expression level of a structural gene is adjusted to achieve the effects of regulating and controlling the real-time expression quantity of D, D-carboxypeptidase and effectively promoting recombinant protein exosecretion. The co-expression system constructed by the invention can improve the permeability of the outer membrane of the recombinant escherichia coli, improves the extracellular secretion level of the recombinant protein, and has important guiding significance for efficient secretion and production of the recombinant protein in the escherichia coli.

Description

technical field [0001] The invention relates to a method and application for accurately and quantitatively regulating the extracellular secretion level of Escherichia coli recombinant protein, and belongs to the fields of genetic engineering and fermentation engineering. Background technique [0002] Escherichia coli expression system is the most commonly used recombinant protein expression system in genetic engineering. Escherichia coli occupies an important position in gene expression technology and is an important tool in the process of molecular biology research and biotechnology industrialization. It is one of the commonly used hosts for efficient production of recombinant proteins, but in the heterologous expression of E. coli, most of the recombinant proteins are secreted into the periplasmic space under the guidance of signal peptides, which will lead to the accumulation of a large number of intermediates, which will affect the production of proteins. hinder. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N9/88C12N15/60C12N1/21C12P21/00A23L29/00A23J1/00C12R1/19
CPCC12N15/70C12N9/88C12Y402/0102C12N15/52C12N1/20C12P21/00A23L29/065A23L29/00A23J1/008C12N2800/60Y02A50/30
Inventor 陈献忠杨海泉官剑民沈微夏媛媛曹钰
Owner JIANGNAN UNIV
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