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Fermentation culture method of recombinant escherichia coli for producing insulin aspart fusion protein

A technology for recombining Escherichia coli and insulin aspart, which is applied in the direction of insulin, microbial-based methods, fermentation, etc., can solve the problems of low expression, long induction culture time, and low production efficiency

Inactive Publication Date: 2022-01-28
NINGBO KUNPENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the fermentation method of the insulin aspart fusion protein in the prior art still has problems such as low expression level, too long induction culture time, and low production efficiency. There is an urgent need to develop new recombinant bacteria producing the insulin aspart fusion protein in this field. Fermentation culture method

Method used

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  • Fermentation culture method of recombinant escherichia coli for producing insulin aspart fusion protein
  • Fermentation culture method of recombinant escherichia coli for producing insulin aspart fusion protein
  • Fermentation culture method of recombinant escherichia coli for producing insulin aspart fusion protein

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preparation example Construction

[0087] Preparation of seed liquid: Inoculate the preserved recombinant Escherichia coli glycerol tube strain in a 500mL shake flask containing 100mL LB seed recovery medium, culture on a shaking table at 37±0.5°C, 220±20rpm for 8.5-10.5h, OD 600 After reaching 1.05-4.30, it is used as seed resuscitation solution; connect 1mL seed resuscitation solution to a 500mL shake flask containing 100mL seed adaptive growth medium, culture at 37±0.5°C, 220±20rpm shaker for 5.5-6.5h, OD 600 Reach 2.5-10.5 as seed liquid for fermentation.

[0088] Batch culture: 0.1L seed liquid was put into a 5L fermenter containing 2.0L fermentation medium, the initial culture conditions were 33°C, pH 7.0, rotation speed 200rpm, ventilation rate 1vvm, tank pressure 0.03mPa. As the cultivation progresses, increase the rotational speed and air flow accordingly, so that the dissolved oxygen is controlled at 5-40%, and cultivate for 4-5 hours. After the dissolved oxygen and pH rise rapidly, enter the fed-batc...

Embodiment 1

[0100]Preparation of seed liquid: Inoculate the preserved recombinant Escherichia coli glycerol tube strain in a 500mL shake flask containing 100mL LB seed recovery medium, culture on a shaking table at 37±0.5°C, 220±20rpm for 8.5-10.5h, OD 600 After reaching 1.05-4.30, it is used as seed resuscitation solution; connect 1mL seed resuscitation solution to a 500mL shake flask containing 100mL seed adaptive growth medium, culture at 37±0.5°C, 220±20rpm shaker for 5.5-6.5h, OD 600 Reach 2.5-10.5 as seed liquid for fermentation;

[0101] Batch culture: 0.1L seed liquid was put into a 5L fermenter containing 2.0L fermentation medium, the initial culture conditions were 33°C, pH 7.0, rotation speed 200rpm, ventilation rate 1vvm, tank pressure 0.03mPa. As the cultivation progresses, increase the rotation speed and air flow accordingly, so that the dissolved oxygen is controlled at 5-40%, and cultivate for 4-5 hours. After the dissolved oxygen and pH rise rapidly, enter the feeding fer...

Embodiment 2

[0108] Preparation of seed liquid: Inoculate the preserved recombinant Escherichia coli glycerol tube strain in a 500mL shake flask containing 100mL LB seed recovery medium, culture on a shaking table at 37±0.5°C, 220±20rpm for 8.5-10.5h, OD 600 After reaching 1.05-4.30, it is used as seed resuscitation solution; connect 1mL seed resuscitation solution to a 500mL shake flask containing 100mL seed adaptive growth medium, culture at 37±0.5°C, 220±20rpm shaker for 5.5-6.5h, OD 600 Reach 2.5-10.5 as seed liquid for fermentation;

[0109] Batch culture: 0.1L seed liquid was put into a 5L fermenter containing 2.0L fermentation medium, the initial culture conditions were 33°C, pH 7.0, rotation speed 200rpm, ventilation rate 1vvm, tank pressure 0.03mPa. As the cultivation progresses, increase the rotation speed and air flow accordingly, so that the dissolved oxygen is controlled at 5-40%, and cultivate for 4-5 hours. After the dissolved oxygen and pH rise rapidly, enter the feeding fe...

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Abstract

The invention provides a culture medium suitable for fermenting recombinant escherichia coli to produce insulin aspart fusion protein. Specifically, the invention provides the culture medium containing yeast peptone, yeast extract powder, glycerol, Boc-L-lysine, a buffering agent and trace elements. The invention further provides a method for producing the insulin aspart fusion protein by fermentation of the culture medium. When the culture medium is used for fermentation, high bacterial density and high insulin aspart fusion protein yield can be obtained, the fermentation culture time is short, raw materials are easy to obtain, the virus carrying risk is avoided, the cost is low, and the culture medium is suitable for industrial production.

Description

technical field [0001] The invention relates to the field of microbial fermentation, and more specifically relates to a recombinant Escherichia coli fermentation culture method for producing insulin aspart fusion protein. Background technique [0002] Diabetes is a common endocrine and metabolic disease. There are currently an estimated 463 million adults (aged 20-79) suffering from diabetes worldwide, accounting for 9.3% of the world's total population in this age group. By 2030 and 2045, this number is expected to reach 578 million (10.2%) and 700 million (10.9%) respectively. my country currently has 116.4 million (not included in Hong Kong, Macao and Taiwan data) diabetic patients, ranking first in the world. Diabetes has become another serious chronic disease after cardiovascular and cerebrovascular diseases and tumors. Diabetes can be divided into three types: type 1, type 2 and pregnancy type. . [0003] Insulin has been regarded as an important drug for treating d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P21/02C12R1/19
CPCC12N1/20C07K14/62C07K2319/60C07K2319/50C07K2319/02
Inventor 李克朗张振山吴松刘慧玲
Owner NINGBO KUNPENG BIOTECH CO LTD
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