Method for fermenting recombinant protein by micro-aerobic induction of escherichia coli

A technology of Escherichia coli and protein, applied in the field of recombinant protein biological products, can solve the problems of low price, excessive protein expression, low induction strength, etc., achieve the effect of reducing requirements, simplifying fermentation equipment, and facilitating later amplification

Pending Publication Date: 2022-08-05
可孚医疗科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

IPTG (isopropyl-β-D-thiogalactopyranoside) and lactose are commonly used as inducers to induce the expression of target proteins in Escherichia coli. However, IPTG has potential toxicity to the human body, and there may be some unsafe factors. At the same time, its strong induction effect may cause excessive protein expression and the formation of inclusion bodies; and lactose, as a natural inducer, can pass through Under the action of the enzyme, it enters the cell and transforms into an allolactose-induced operon. It has the advantages of low price, non-toxicity, suitable for industrial scale-up production, and can also be used as a carbon source to promote cell growth, low induction intensity, and promote protein soluble expression.

Method used

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  • Method for fermenting recombinant protein by micro-aerobic induction of escherichia coli

Examples

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Effect test

Embodiment 1

[0036] (1) Construction of strains

[0037] Using Escherichia coli BL21(DE3) strain, 6 histidine tags were introduced at the end of the human-like collagen protein gene, and then connected to the vector pET-24a(+) to construct the expression plasmid col-pET-24a(+), which will be verified correctly The recombinant plasmid was transformed into E. coli competent cells BL21 (DE3), and the genetically engineered bacteria E expressing recombinant human-like collagen were all positive for LB plate identification containing kanamycin, colony PCR identification and sequencing identification. The .coil BL21(DE3) / pET-24a(+)-col strain was stored in a -80°C refrigerator until use.

[0038]Specifically, the recombinant human-like collagen base sequence of the above-mentioned recombinant human-like collagen genetically engineered bacteria is artificially composed of an eight-repeat type I human collagen sequence fragment and a type III human collagen sequence fragment. Splicing synthesis (...

Embodiment 2

[0059] (1) Construction of strains

[0060] Using Escherichia coli BL21(DE3) strain, 6 histidine tags were introduced at the end of the collagen gene, and then connected to the vector pET-24a(+) to construct the expression plasmid col-pET-24a(+), and the correct recombinant plasmid will be verified. It was transformed into E. coli competent cells BL21 (DE3), and the genetically engineered bacteria E.coil BL21 expressing recombinant human collagen was positive for the identification of LB plate containing kanamycin, colony PCR identification and sequencing identification. (DE3) / pET-24a(+)-col strain was stored in a -80°C refrigerator until use.

[0061] Specifically, the recombinant human-like collagen base sequence of the above-mentioned recombinant human-like collagen genetically engineered bacteria is artificially composed of an eight-repeat type I human collagen sequence fragment and a type III human collagen sequence fragment. Splicing synthesis (refer to the prior art fo...

Embodiment 3

[0082] (1) Construction of strains

[0083] Using Escherichia coli BL21(DE3) strain, 6 histidine tags were introduced at the end of the collagen gene, and then connected to the vector pET-24a(+) to construct the expression plasmid col-pET-24a(+), and the correct recombinant plasmid will be verified. It was transformed into E. coli competent cells BL21 (DE3), and the genetically engineered bacteria E.coil BL21 expressing recombinant human collagen was positive for the identification of LB plate containing kanamycin, colony PCR identification and sequencing identification. (DE3) / pET-24a(+)-col strain was stored in a -80°C refrigerator until use.

[0084]Specifically, the recombinant human-like collagen base sequence of the above-mentioned recombinant human-like collagen genetically engineered bacteria is artificially composed of an eight-repeat type I human collagen sequence fragment and a type III human collagen sequence fragment. Splicing synthesis (refer to the prior art for...

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Abstract

The invention provides a method for fermenting recombinant human-like collagen by micro-aerobic induction of escherichia coli, which comprises the steps of strain activation, seed solution culture and fed-batch fermentation, and is characterized in that the fed-batch fermentation process comprises the following steps: a cell rapid propagation stage: inoculating a secondary seed solution into a basic fermentation culture medium for culture; in the culture process, the dissolved oxygen is maintained to be 30-33%, and the specific growth rate of the genetically engineered bacteria is controlled to be 0.1-0.5 by supplementing a first supplementing culture medium into the fermentation liquid; a micro-aerobic induced expression recombinant protein stage: feeding lactose into the fermentation liquid to induce gene engineering bacteria to express protein, and replacing the first fed-batch culture medium with a second fed-batch culture medium; feeding back and feeding a lactose inducer according to the dissolved oxygen content in the fermentation liquid, and maintaining the dissolved oxygen at 0-5% until the fermentation is finished.

Description

technical field [0001] The invention relates to the technical field of recombinant protein biological products, in particular to a method for microaerobic induction of Escherichia coli to ferment recombinant proteins. Background technique [0002] Escherichia coli is a commonly used host bacteria. Its genome, metabolome and proteome have been extensively studied by researchers and are often used to produce recombinant proteins and enzymes. The recombinant proteins expressed by Escherichia coli have the following characteristics: easy growth control, for bacterial culture The material is less expensive than that of mammalian cell systems, lacks post-translational processing such as modification and glycosylation, phosphorylation, and often forms inclusion bodies, which affect the biological activity and conformation of the expressed protein. IPTG (isopropyl-β-D-thiogalactoside) and lactose are commonly used as inducers for the induction and expression of target proteins in Es...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K14/78C12N1/21C12R1/19
CPCC12P21/02C07K14/78
Inventor 黄琪黄微景成宇廖经练刘丽霞
Owner 可孚医疗科技股份有限公司
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