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a reinforcement mut s Method for expressing heterologous protein in Pichia pastoris

A Pichia pastoris and heterologous protein technology, applied in the field of fermentation engineering, can solve the problems of methanol safety hazards, large safety hazards and high operating costs, and achieve the effects of eliminating operational safety hazards, increasing consumption speed, and improving concentration and activity.

Active Publication Date: 2020-12-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, while utilizing pure O 2 Although methanol and methanol can increase the expression level of the target heterologous protein, there are high operating costs and great safety hazards (while using pure O 2 and methanol have huge potential safety hazards) and many other problems

Method used

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  • a reinforcement mut <sup>s</sup> Method for expressing heterologous protein in Pichia pastoris
  • a reinforcement mut <sup>s</sup> Method for expressing heterologous protein in Pichia pastoris
  • a reinforcement mut <sup>s</sup> Method for expressing heterologous protein in Pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1: Expression performance of porcine interferon under the methanol cycle induction control strategy

[0094] Specific steps are as follows:

[0095](1) The fed-batch culture of Pichia pastoris was carried out in a 5L fermenter, and the liquid volume of the initial fermentation medium was 2.3L. The methanol carrying the exogenous porcine interferon-α gene was used in the initial fermentation of the slow recombinant Pichiapastoris KM71 strain. The inoculum size in the medium is 13% (V / V);

[0096] (2) After the cultivation starts, the ventilation rate is controlled at 3vvm (air), and the stirring speed is increased to maintain the DO in the fermented liquid above 10% all the time for cultivation;

[0097] (3) When the glycerol in the initial medium was exhausted, the improved DO-Stat control strategy was adopted (Ding J, Gao M J, Hou G L, et al. Stabilizing porcine interferon-αproduction by Pichiapastoris with an ethanol on ‐line measurement based DO ‐Stat glyce...

Embodiment 2

[0113] Example 2: Expression performance of human lysozyme under the control strategy of methanol cycle induction

[0114] The methanol utilization slow-type recombinant Pichiapastoris KM71 strain carrying the exogenous porcine interferon alpha gene in Example 1 was replaced with the methanol utilization slow-type recombinant Pichiapastoris KM71 strain carrying the exogenous lysozyme (human lysozyme, hLZY) gene, and the induction time was 71h (fermentation performance and main parameters are shown in Table 1).

[0115] It can be seen from Table 1 that the final concentration of human lysozyme in the fermentation broth is 1.46g / L, and the enzyme activity of human lysozyme is 2.15×10 5 IU / mL.

[0116] In order to verify the invention patent (application) "a kind of enhanced Mut S The versatility of the method of Pichia pastoris expressing heterologous protein ", three kinds of methanol induction methods (comparative example 1, comparative example 2, embodiment 1) that are use...

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Abstract

The invention discloses a method for intensifying expression of heterologous protein by MutS type pichia pastoris, and belongs to the technical field of fermentation engineering. According to the method disclosed by the invention, the methanol concentration and the dissolved oxygen (DO) concentration in a methanol induction process of MutS type pichia pastoris are switched back and forth between high / low level and low / high level according to different cycle lengths so as to improve the metabolic activity of the MutS type pichia pastoris and the specific consumption speed of methanol, reduce the accumulation of intracellular methanol and remarkably improve the concentration and activity of the heterologous protein; by adopting the method, the expression level of heterologous protein by theMutS type pichia pastoris is increased by about 1.5 times over traditional high methanol concentration / low DO induction strategy and relatively low methanol concentration / high DO induction strategy.

Description

technical field [0001] The present invention relates to a kind of strengthening Mut S The invention discloses a method for expressing heterologous proteins in Pichia pastoris, belonging to the technical field of fermentation engineering. Background technique [0002] Controlling the fixed value of state variables (such as temperature, dissolved oxygen concentration, cell specific growth rate, substrate specific consumption rate, etc.) at a constant level is the most common control method to optimize the microbial fermentation process, and is widely used in Pichia pastoris , Saccharomyces cerevisiae, Escherichia coli and Bacillus subtilis in the fermentation production process. [0003] Methylotrophic Pichia pastoris (Methylotrophic Pichia pastoris) is widely used in the expression of heterologous proteins, and plays an important role in the production of food, medicine, etc. The culture / induction process is controlled by fixed value to increase the expression of heterologo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/38C12P21/00C12R1/84
CPCC12N1/38C12P21/00
Inventor 丁健史仲平李程程贾禄强严建
Owner JIANGNAN UNIV
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