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Method for preparing semeglutide precursor through high-density fermentation

A technology of high-density fermentation and semaglutide, which is applied in the field of high-density fermentation, can solve the problem that the fermentation expression needs to be improved, and achieves the effects of increasing the bacterial fermentation expression, reducing production costs, and having broad industrial application prospects.

Active Publication Date: 2021-10-15
BEIJING HUIZHIHENG BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method needs to use a variety of specific components, and the expression level of fermentation still needs to be improved

Method used

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  • Method for preparing semeglutide precursor through high-density fermentation
  • Method for preparing semeglutide precursor through high-density fermentation
  • Method for preparing semeglutide precursor through high-density fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of recombinant Escherichia coli engineering bacteria that stably and highly express semaglutide precursor

[0054] (1) Synthesize the fusion polypeptide encoding genes shown in SEQ ID No.7 and SEQ ID No.8 respectively, and add Nde I enzyme cutting site, 3' end add stop codon and xho I restriction site;

[0055] (2) The gene fragments prepared in step (1) were respectively cloned into the expression vector pET-30a(+) by enzyme digestion. Nde I and xho Between the I enzyme cutting sites, thereby constructing two kinds of recombinant expression vectors pET-30a(+)-A1-GLP-1 and pET-30a(+)-A2-GLP-1;

[0056] (3) Transform the recombinant expression vectors obtained in step (2) into Escherichia coli BL21(DE3) by heat shock method, and select 8 clones after resistance screening, and select 8 clones from the two recombinant engineering bacteria, and name them respectively A1-GLP-1-1 to A1-GLP-1-8 and A2-GLP-1-1 to A2-GLP-1-8 were stored in glycerol....

Embodiment 2

[0058] Embodiment 2: the high-density fermentation (low Mg of recombinant escherichia coli engineering bacterium) 2+ concentration and low OD 600 induced value)

[0059] (1) Solution preparation

[0060] A. Ammonia preparation

[0061] Take 300 ml of purified water in a feeding bottle, and after sterilizing at 121 °C for 30 min, add an equal volume of ammonia water into the feeding bottle under aseptic conditions and mix well for use.

[0062] B. activation medium

[0063] The composition of the activation medium was tryptone 20g / L, yeast extract powder 10g / L, sodium chloride 10g / L; sterilized at 121°C for 30min.

[0064] C. Solution D preparation

[0065] Solution D consists of ferrous sulfate heptahydrate 3.36g / L, zinc sulfate heptahydrate 0.84g / L, manganese sulfate monohydrate 0.51g / L, ammonium molybdate tetrahydrate 0.18g / L, copper sulfate pentahydrate (II) 0.12g / L, phosphoric acid 48ml / L; 0.22μm filter filter sterilization.

[0066] D. feed medium

[0067] 30-...

Embodiment 3

[0079] Embodiment 3: the high-density fermentation of recombinant escherichia coli engineering bacteria

[0080] (1) Solution preparation

[0081] A. Ammonia preparation

[0082] Take 300 ml of purified water in a feeding bottle, and after sterilizing at 121 °C for 30 min, add an equal volume of ammonia water into the feeding bottle under aseptic conditions and mix well for use.

[0083] B. activation medium

[0084] The medium consists of tryptone 20g / L, yeast extract powder 10g / L, sodium chloride 10g / L; sterilized at 121°C for 30 min.

[0085] C. Solution D preparation

[0086] Solution D consists of ferrous sulfate heptahydrate 3.36g / L, zinc sulfate heptahydrate 0.84g / L, manganese sulfate monohydrate 0.51g / L, ammonium molybdate tetrahydrate 0.18g / L, copper sulfate pentahydrate (II) 0.12g / L, phosphoric acid 48ml / L; 0.22μm filter filter sterilization.

[0087] D. feed medium

[0088] 30-50% glucose and 5-25% yeast extract, the specific concentration, supplementation...

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Abstract

The invention discloses a method for producing a simeglutide precursor by high-density fermentation of recombinant escherichia coli. In order to solve the problem that the expression quantity of semeglutide produced through fermentation at present is low, the Mg ion concentration is remarkably increased in a high-density fermentation culture medium of prepared recombinant escherichia coli for expressing a semeglutide precursor, the induction OD value of fermentation culture is increased to 150 or above, the fermentation OD value of the recombinant escherichia coli and the thallus biomass of inclusion bodies are remarkably increased, the high-density fermentation effect of the recombinant engineering bacteria is realized, and the expression quantity of a semeglutide precursor is obviously increased.

Description

technical field [0001] The invention belongs to the technical field of microbial engineering, and in particular relates to a high-density fermentation method of recombinant engineering bacteria expressing semaglutide precursors. Background technique [0002] With the development of social economy and the gradual improvement of people's living standards, great changes have taken place in people's diet structure, which has also led to an increase in the incidence of obesity, and further led to a sharp increase in the number of diabetic patients. According to statistics, the number of diabetic patients in my country exceeds 100 million, and there are more than 150 million hidden pre-diabetic patients. Diabetes has become the third leading chronic disease. [0003] Diabetes is a complex chronic metabolic disease caused by the long-term interaction of genetic and environmental factors. It is caused by the lack of insulin secretion and is characterized by hyperglycemia. It is div...

Claims

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Application Information

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IPC IPC(8): C12P21/02C12N1/21C12N15/70C07K14/605C12R1/19
CPCC12P21/02C07K14/605C12N15/70
Inventor 曹海燕连婕妮林兆生朱志伟王惠王洪宇
Owner BEIJING HUIZHIHENG BIOTECHNOLOGY CO LTD
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