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Method for improving expression level of Pichia pastoris recombinant protein

A recombinant protein, Pichia pastoris technology, applied in the field of bioengineering, can solve the problems affecting the expression of exogenous proteins, yeast cell free radical accumulation, yeast death and other problems, and achieve the effect of overcoming free radical accumulation.

Inactive Publication Date: 2011-07-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of high-density fermentation, various conditions are difficult to control, and the accumulation of free radicals inside the yeast cells is serious, which easily leads to the death of the yeast, which in turn affects the expression of foreign proteins

Method used

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  • Method for improving expression level of Pichia pastoris recombinant protein
  • Method for improving expression level of Pichia pastoris recombinant protein

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Experimental program
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Effect test

Embodiment 1

[0014] The engineered bacteria GS115 / pPIC9K-IL2-HSA expressing interleukin-2-human serum albumin fusion protein was constructed and preserved in our laboratory, and the methanol utilization phenotype of the recombinant Pichia pastoris was mut + .

[0015] (1) Pick a single colony from the plate where the high-expression strain is preserved, place it in a 50 mL shake flask containing 10 mL of LYPD medium, and culture it at 30° C. and 200 rpm for 24 hours. (2) The bacteria cultured in YPD medium for 24 h were inoculated into a 1000 mL shake flask containing 200 mL of BMGY medium at a ratio of 1:100, and cultured at 30° C. and 200 rpm for 48 h. During this period, 1 mL of bacterial suspension was taken every 4 hours, and the bacterial concentration was detected by ultraviolet spectrophotometer (bacterial concentration is expressed as A 600 is the counting unit), and draw the growth curve.

[0016] (3) Repeat steps (1) and (2). When the bacteria grow to 36 hours, put the bacteri...

Embodiment 2

[0018] The engineered bacteria GS115 / pPIC9K-IFNβ-HSA expressing β-interferon-human serum albumin fusion protein was constructed and preserved in our laboratory, and the methanol utilization phenotype of the recombinant Pichia pastoris was mut + .

[0019] (1) Pick a single colony from the plate where the high-expression strain is preserved, place it in a 50 mL shake flask containing 10 mL of LYPD medium, and culture it at 30° C. and 200 rpm for 24 hours. (2) The bacteria cultured in YPD medium for 24 h were inoculated into a 1000 mL shake flask containing 200 mL of BMGY medium at a ratio of 1:100, and cultured at 30° C. and 200 rpm for 48 h. During this period, 1 mL of bacterial suspension was taken every 4 hours, and the bacterial concentration was detected by ultraviolet spectrophotometer (bacterial concentration is expressed as A 600 is the counting unit), and draw the growth curve.

[0020] (3) Repeat steps (1) and (2). When the bacteria grow to 36 hours, put the bacteri...

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Abstract

The invention relates to a method for improving the expression level of Pichia pastoris recombinant protein. On the basis of the normal process flow of recombinant Pichia pastoris, trace melatonin is added to greatly improve the expression of the recombinant protein. By utilizing the method, radical accumulation in yeast brought by intimidation, such as unbalanced oxygen supply, during the high-density fermentation of the recombinant saccharomycete is lowered, and the damage on the yeast brought by the radical in the high-density fermentation process is eliminated so as to improve the physiological condition of the yeast. After the melatonin is used, the expression level of the recombinant protein is improved to a large extent.

Description

technical field [0001] The invention relates to a new method for improving the expression of recombinant protein, which belongs to the field of bioengineering. Background technique [0002] Pichia pastoris expression system is currently one of the best and most widely used exogenous gene expression systems. As a eukaryotic expression system, the Pichia pastoris expression system has many advantages that other protein expression systems do not have. The expressed protein can be processed, folded and post-translationally modified, so that the expressed protein has biological activity, has a strong alcohol oxidase (AOX1) gene promoter, can strictly regulate the expression of foreign proteins, and has low nutritional requirements. The culture medium is cheap, grows fast, and is easy to operate and culture compared with higher eukaryotic cells such as insects and mammals. Pichia pastoris has a strong preference for aerobic growth. This physiological characteristic enables it to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/38C12N1/19C12R1/85
Inventor 朱瑞宇金坚辛鑫张大成雷楗勇陈蕴
Owner JIANGNAN UNIV
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