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Method for high-density fermentation of pichia pastoris of insulin precurosor protein

An insulin precursor, high-density fermentation technology, applied in the field of microorganisms, can solve the problems of complex operation and high cost, and achieve the effects of simple process, low cost and convenient operation

Active Publication Date: 2017-01-04
YICHANG HEC CHANGJIANG PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can effectively reduce O-glycosylation, the method is costly and complicated

Method used

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  • Method for high-density fermentation of pichia pastoris of insulin precurosor protein
  • Method for high-density fermentation of pichia pastoris of insulin precurosor protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Object: GS115 Pichia pastoris (purchased from Invitrogen, USA) was used to ferment and express recombinant human insulin precursor

[0033] Strain expansion: inoculate the activated strains into the seed medium, shake and cultivate at 28°C for 10 hours, then transfer to the secondary seed medium at a volume ratio of 0.5%, and shake and cultivate at 28°C for 20 hours;

[0034]Fermentation culture: 1) Batch growth stage: inoculate the strains after the expansion into the fermentation medium, the inoculation volume of the strains is 2.5% of the volume of the fermentation medium, and the dissolved oxygen is controlled by the rotating speed to be greater than or equal to 50%. Cultivate at ℃ until the dissolved oxygen rebounds to 80%; 2) Glycerin supplementation stage: supplement glycerin solution at a rate of 25g / L / h, control dissolved oxygen ≥ 30% by rotating speed, and add urea to control the pH value at 4.0, Cultivate until the cell concentration is OD 600 =200, stop add...

Embodiment 2

[0038] Object: Pichia pastoris GS115 (purchased from Invitrogen, USA) was used to ferment and express recombinant human insulin precursor.

[0039] Strain expansion: inoculate the activated strains into the seed medium, shake and culture at 32°C for 14 hours, then transfer to the secondary seed medium at a volume ratio of 1.5%, and shake and cultivate at 32°C for 24 hours;

[0040] Fermentation culture: 1) Batch growth stage: inoculate the strains after the expansion into the fermentation medium, the inoculation volume of the strains is 2.5% of the volume of the fermentation medium, control the dissolved oxygen ≥ 50% by rotating speed, at 32 Cultivate at ℃ until the dissolved oxygen rebounds to 80%; 2) Glycerin supplementation stage: supplement glycerol solution at a rate of 18g / L / h, control dissolved oxygen ≥ 30% by rotating speed, and add urea to control the pH value at 5.0, Cultivate until the cell concentration is OD 600 =300, stop adding glycerin; 3) Methanol induction s...

Embodiment 3

[0044] Object: Pichia pastoris GS115 (purchased from Invitrogen, USA) was used to ferment and express recombinant human insulin precursor.

[0045] Strain expansion: Inoculate the activated strains into the seed medium, shake and culture at 30°C for 10-14 hours, then transfer to the secondary seed medium at a volume ratio of 1.0%, and shake and culture at 30°C for 22 hours ;

[0046] Fermentation culture: 1) Batch growth stage: inoculate the strains after the expansion into the fermentation medium, the inoculation volume of the strains is 2.5% of the volume of the fermentation medium, control the dissolved oxygen ≥ 50% by rotating speed, at 30 Cultivate at ℃ until the dissolved oxygen rebounds to 80%; 2) Glycerol supplementation stage: supplement glycerin solution at a rate of 20g / L / h, control dissolved oxygen ≥ 30% by rotating speed, and add urea to control the pH value at 5.0, Cultivate until the cell concentration is OD 600 Stop adding glycerin at 250 ℃; 3) Methanol induc...

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Abstract

The invention relates to the field of microorganisms and discloses a method for high-density fermentation of pichia pastoris of insulin precurosor protein. The method comprises the steps of expanding culture of strain, fermentation culture and thallus collection. Fermentation culture is divided into a batch growth phase, a glycerinum replenishment phase and a methyl alcohol induction phase. By controlling parameters including age, replenishing rate and pH value of strain during fermentation, the concentration of glycosylation 2 can be effectively reduced by 60+ / -8%, and the unit yield of insulin precurosor protein can be increased by 25% or above. Furthermore, the method has the advantages of being simple, easy to operate, low in cost and the like.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a high-density fermentation method of Pichia pastoris for insulin precursor protein. Background technique [0002] Currently, various microbial expression systems have been used to produce insulin and its derivatives, such as Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, etc. Due to the disadvantages of low expression level and difficult purification, the application of the first two systems is less. Pichia pastoris has the advantages of high-efficiency expression and secretion of extracellular proteins, and low production costs, so it is widely used in the commercial production of recombinant proteins. [0003] However, Pichia pastoris inevitably undergoes post-translational modification of proteins during the process of expressing foreign proteins, which are then present as impurities in the final product, which is difficult to purify. Of these, the most common fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C12R1/84
CPCC07K14/62C12P21/00
Inventor 贾永峰宁荣良王进军黄成潭张奕敏张宏达
Owner YICHANG HEC CHANGJIANG PHARMA CO LTD
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