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Pichia pastoris fermentation medium for large scale production of recombinant human collagen

A technology of human collagen and fermentation medium, applied in the direction of microorganism-based methods, fermentation, microorganisms, etc., can solve the problems of high skill requirements for workers, difficult operation, and high work intensity, so as to reduce production costs and reduce energy consumption. Consumption, the effect of shortening the induction cycle

Active Publication Date: 2016-12-28
浙江诸暨聚源生物技术有限公司
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0005] In order to solve the actual production problems in the large-scale production of recombinant protein expressed by high-density fermentation of Pichia pastoris, such as long time consumption, high energy consumption, difficult operation, high work intensity, and high skill requirements for workers, the present invention provides a method suitable for Pichia pastoris fermentation medium for large-scale production of recombinant human collagen. The fermentation medium contains metabolic regulators, which can effectively regulate the metabolism of Pichia pastoris and accelerate the expression of recombinant proteins. It is suitable for industrial fermentation and cultivation of Pichia pastoris. Large-Scale Production of Recombinant Human Collagen

Method used

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  • Pichia pastoris fermentation medium for large scale production of recombinant human collagen
  • Pichia pastoris fermentation medium for large scale production of recombinant human collagen
  • Pichia pastoris fermentation medium for large scale production of recombinant human collagen

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Fermentation medium: 85% H 3 PO 4 26.7mL / L; CaSO 4 2H 2 O 1.175g / L; K 2 SO 4 18.2g / L; MgSO 4 ·7H 2 O 14.9g / L; KOH 4.13g / L; glycerin 40.0g / L; PTM1 4.35mL / L; pyruvate 0.01g / L.

[0020] Add the seed solution to a 1000L fermenter containing 500L fermentation medium according to 10% inoculation amount, adjust the stirring speed to 200-600rpm, the tank pressure to 0.2-1.0bar, and adjust the air flow to make dissolved oxygen (DO) > 30%. When the carbon source was exhausted, the dissolved oxygen rose sharply, and 50% glycerin was added to replenish the carbon source until the wet weight of the bacteria was 215g / L, and the 50% glycerol was stopped. After the glycerol is exhausted, add methanol as a new carbon source, enter the methanol induction culture stage, and adjust the rotation speed, tank pressure, air flow, and methanol feeding speed to make the dissolved oxygen (DO) > 20%, and take samples every 8 hours , measure the wet weight of the bacteria and the expressi...

Embodiment 2

[0022] Fermentation medium: 85% H 3 PO 4 26.7mL / L; CaSO 4 2H 2 O 1.175g / L; K 2 SO 4 18.2g / L; MgSO 4 ·7H 2 O 14.9g / L; KOH 4.13g / L; glycerin 40.0g / L; PTM1 4.35mL / L; pyruvate 10g / L.

[0023] Add the seed solution to a 1000L fermenter containing 500L fermentation medium according to 10% inoculation amount, adjust the stirring speed to 200-600rpm, the tank pressure to 0.2-1.0bar, and adjust the air flow to make dissolved oxygen (DO) > 30%. When the carbon source was exhausted, the dissolved oxygen rose sharply, and 50% glycerol was added to supplement the carbon source until the wet weight of the bacteria was 216g / L, and the 50% glycerol was stopped. After the glycerol is exhausted, add methanol as a new carbon source, enter the methanol induction culture stage, and adjust the rotation speed, tank pressure, air flow, and methanol feeding speed to make the dissolved oxygen (DO) > 20%, and take samples every 8 hours , measure the wet weight of the bacteria and the expressio...

Embodiment 3

[0025] Fermentation medium: 85% H 3 PO 4 26.7mL / L; CaSO 4 2H 2 O 1.175g / L; K 2 SO 4 18.2g / L; MgSO 4 ·7H 2 O 14.9g / L; KOH 4.13g / L; glycerin 40.0g / L; PTM1 4.35mL / L; pyruvate 0.1g / L.

[0026] Add the seed solution to a 1000L fermenter containing 500L fermentation medium according to 10% inoculation amount, adjust the stirring speed to 200-600rpm, the tank pressure to 0.2-1.0bar, and adjust the air flow to make dissolved oxygen (DO) > 30%. When the carbon source was exhausted, the dissolved oxygen rose sharply, and 50% glycerol was started to supplement the carbon source until the wet weight of the bacteria was 218g / L, and the 50% glycerol was stopped. After the glycerol is exhausted, add methanol as a new carbon source, enter the methanol induction culture stage, and adjust the rotation speed, tank pressure, air flow, and methanol feeding speed to make the dissolved oxygen (DO) > 20%, and take samples every 8 hours , measure the wet weight of the bacteria and the expres...

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Abstract

The invention discloses a pichia pastoris fermentation medium for large scale production of recombinant human collagen. 0.01-10g / L of a metabolism conditioning agent is used in the medium so that pichia pastoris metabolism is adjusted, recombinant protein expression is accelerated, a fermentation period is shortened, energy consumption is reduced, an error risk is reduced and stable industrial production is guaranteed. The medium realizes large scale production of recombinant human collagen through 1000L of a fermentation cylinder, shortens a methanol induction period by 20% and improves a recombinant human collagen expression level by 16.5%. The pichia pastoris fermentation medium is especially suitable for a pichia pastoris methanol induction stage of recombinant human collagen industrial high density fermentation expression, effectively reduces a production cost, produces substantial economic benefits and has a very important meaning for industrial production of the recombinant human collagen.

Description

technical field [0001] The invention belongs to the field of high-density fermentation and expression of recombinant protein by Pichia pastoris, and relates to a Pichia fermentation medium suitable for large-scale production of recombinant human collagen, more specifically, relates to a high-density fermentation expression suitable for industrial scale Fermentation medium for the methanol-induced phase of Pichia pastoris with recombinant human collagen. Background technique [0002] As a new type of yeast expression system, the Pichia pastoris expression system has both the characteristics of prokaryotic microorganisms and the characteristics of eukaryotic organisms. It can perform post-translational modifications such as glycosylation and disulfide bond formation on the target protein, and it is becoming more and more common. It can be used instead of Escherichia coli to realize genetic manipulation and express functional active protein. So far, more than 1,000 foreign pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12R1/84
CPCC12P21/02C12N1/165C12R2001/84
Inventor 杨树林高力虎黄建民杜尔凤赵健烽陶海冯丽萍周爱梅季乐
Owner 浙江诸暨聚源生物技术有限公司
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