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Method for improving escherichia coli protein expression quantity through staged dissolved oxygen control

A technology of protein expression and Escherichia coli, which is applied in the field of fermentation engineering, can solve the problems of decreased cell viability, protein degradation, cell aging, etc., to increase expression, slow cell aging and autolysis, and maintain cell vitality Effect

Pending Publication Date: 2022-06-28
江苏万邦医药科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the later stage of induction, bacterial aging and autolysis are prone to occur, bacterial viability decreases, and protein degradation occurs, which limits the further improvement of protein expression.

Method used

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  • Method for improving escherichia coli protein expression quantity through staged dissolved oxygen control
  • Method for improving escherichia coli protein expression quantity through staged dissolved oxygen control
  • Method for improving escherichia coli protein expression quantity through staged dissolved oxygen control

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 The 30L fermentation of genetically engineered bacteria A only includes the first-stage induction;

[0044] 1) First-class seed culture: inoculate the strains into the first-class seed medium, and shake at 30°C and 250rpm for 12-16 hours to obtain first-class seed liquid; the first-class seed medium is LB medium, sodium chloride 10g / L, yeast extract powder 5g / L, peptone 10g / L, sterilized at 121°C for 20min.

[0045] 2) Secondary seed culture: inoculate primary seeds into secondary seed medium, and shake and cultivate at 37°C and 250 rpm for 3 to 6 hours to obtain secondary seed liquid; secondary seed medium is LB medium, 10 g of sodium chloride / L, yeast extract powder 5g / L, peptone 10g / L, sterilized at 121°C for 20min.

[0046] 3) 30L fermentation

[0047] a. Initial culture stage: first prepare 12L fermentation medium, sterilize at 121°C for 20min, cool down to 30°C; adjust pH to 6.7, inoculate 1.2L secondary seeds into the fermenter, the dissolved oxygen...

Embodiment 2

[0057] Example 2 30L fermentation of genetically engineered bacteria A, including first-stage induction and second-stage induction;

[0058] 1) First-class seed culture: inoculate the strains into the first-class seed medium, and shake at 30°C and 250rpm for 12-16 hours to obtain first-class seed liquid; the first-class seed medium is LB medium, sodium chloride 10g / L, yeast extract powder 5g / L, peptone 10g / L, sterilized at 121°C for 20min.

[0059] 2) Secondary seed culture: inoculate primary seeds into secondary seed medium, and shake and cultivate at 37°C and 250 rpm for 3 to 6 hours to obtain secondary seed liquid; secondary seed medium is LB medium, 10 g of sodium chloride / L, yeast extract powder 5g / L, peptone 10g / L, sterilized at 121°C for 20min.

[0060] 3) 30L fermentation

[0061] a. Initial culture stage: first prepare 12L fermentation medium, sterilize at 121°C for 20min, cool down to 30°C; adjust pH to 6.7, inoculate 1.2L secondary seeds into the fermenter, the ...

Embodiment 3

[0072] Example 3 The 30L fermentation of genetically engineered bacteria B only includes the first-stage induction;

[0073] 1) First-class seed culture: inoculate the strains into the first-class seed medium, and shake at 30°C and 250rpm for 12-16 hours to obtain first-class seed liquid; the first-class seed medium is LB medium, sodium chloride 10g / L, yeast extract powder 5g / L, peptone 10g / L, sterilized at 121°C for 20min.

[0074] 2) Secondary seed culture: inoculate primary seeds into secondary seed medium, and shake and cultivate at 37°C and 250 rpm for 3 to 6 hours to obtain secondary seed liquid; secondary seed medium is LB medium, 10 g of sodium chloride / L, yeast extract powder 5g / L, peptone 10g / L, sterilized at 121°C for 20min.

[0075] 3) 30L fermentation

[0076] a. Initial culture stage: first prepare 12L fermentation medium, sterilize at 121°C for 20min, cool down to 30°C; adjust pH to 6.7, inoculate 1.2L secondary seeds into the fermenter, the dissolved oxygen...

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Abstract

The invention discloses a method for improving escherichia coli protein expression quantity through staged dissolved oxygen control. Comprising the steps of strain activation, seed culture and fermentation tank fermentation, and the fermentation tank fermentation is divided into an initial culture stage, a fed-batch growth stage and an induction stage; wherein the induction stage is divided into first-stage induction and second-stage induction; the dissolved oxygen is controlled to be 25% or above through induction in the first stage, and the dissolved oxygen is controlled to be 0-10% through induction in the second stage. According to the method, induction is divided into two stages, and the culture conditions of each stage are controlled, so that high-density fermentation of the escherichia coli genetically engineered bacteria is realized. During the first-stage induction, a fermentation tank is in a maximum oxygen supply state, so that escherichia coli is in a high metabolism state, and rapid expression of protein is realized; during the second-stage induction, the oxygen supply state of the fermentation tank is reduced, so that the supplementing amount of the carbon source and the nitrogen source is reduced, the thalli are in a relatively low metabolism state, the aging and autolysis of the thalli can be slowed down, the vitality of the thalli can be maintained, and further expression of protein is realized.

Description

technical field [0001] The invention belongs to the technical field of fermentation engineering, and in particular relates to a method for increasing the protein expression of Escherichia coli by controlling staged dissolved oxygen. Background technique [0002] In recent years, due to the wide application of recombinant DNA technology and the increasing market demand for drugs and health care products produced by recombinant DNA technology, the combination process of recombinant DNA technology and large-scale culture technology has been accelerated, making it impossible to obtain a large amount. Natural proteins, especially genetically engineered drugs, can be mass-produced for clinical application. Using high-density fermentation technology to increase the fermentation density of the bacteria and ultimately increase the protein expression can not only reduce the culture volume and strengthen the downstream separation and extraction, but also shorten the production cycle an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C12N1/20C12R1/19
CPCC12P21/00C12N1/20
Inventor 文良柱王法兴赵珊珊徐晓峰
Owner 江苏万邦医药科技有限公司
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