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Method for increasing yield of pichia pastoris foreign protein

A technology for exogenous protein and yield, applied in the field of bioengineering, to achieve the effect of good application value and increased expression of exogenous protein

Active Publication Date: 2022-04-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The heat stress response mechanism regulates heat shock proteins and other chaperones to assist in the correct folding of intracellular proteins

Method used

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  • Method for increasing yield of pichia pastoris foreign protein
  • Method for increasing yield of pichia pastoris foreign protein
  • Method for increasing yield of pichia pastoris foreign protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Construction of plasmids overexpressing heat stress-related genes

[0046] Using the Pichia pastoris genome as a template, the P1 and P2 primers were used to amplify the HSP78 gene fragment (the nucleotide sequence is shown in SEQID NO: 1); the vector pGAPZα was used as a template, and the P3 and P4 primers were used to amplify the vector fragment, using The seam cloning kit connects the two fragments into a circular plasmid and immediately transforms it into Escherichia coli competent cells. After sequencing verification, a positive transformant with correct sequencing is obtained, and the plasmid is extracted from the positive transformant to obtain an overexpression plasmid.

[0047] Using the Pichia pastoris genome as a template, primers P5 and P6 amplify the SSA3 gene fragment (nucleotide sequence shown in SEQ ID NO: 2), using the vector pGAPZα as a template, primers P7 and P8 amplify the vector fragment, and use seamless cloning The kit connects the t...

Embodiment 2

[0056] Example 2: Effects of overexpressing HSP78 and SSA3 on the expression of Pichia pastoris green fluorescent protein

[0057] The overexpression plasmid constructed in Example 1 was linearized, and the linearized overexpression plasmid was transformed into P GAP Pichia pastoris expressing green fluorescent protein from the promoter (for the specific construction method of Pichia pastoris, see the literature Oxidativestress tolerance contributions to heterologous protein production in Pichiapastoris, published in 2021), constructing the overexpression of heat stress-related genes in Pichia pastoris Recombinant strains EGFP-SSA3 and EGFP-HSP78. It was determined by RT-qPCR that the gene transcription levels of SSA3 and HSP78 in the overexpression strain were increased by 4.7 times and 6.3 times, respectively, compared with those before the overexpression.

[0058] The constructed recombinant strains EGFP-SSA3 and EGFP-HSP78 were respectively inoculated in YPD medium for ...

Embodiment 3

[0059] Example 3: Verification of the fermentation expression of other foreign proteins by overexpressing HSP78 and SSA3

[0060] By transforming the linearized overexpression plasmids into lipase-expressing yeast respectively (for the specific construction method of Pichia pastoris expressing lipase, see the document Enhancement of lipase r27RCL production in Pichia pastoris by regulating gene dosage and co-expression with chaperoneprotein disulfide isomerase published in 2013 For the strain SRCL1) and phospholipase (for the specific construction method of Pichia expressing phospholipase, refer to the recombinant Pichia GS115 in the literature Streptomyces violeter Phospholipase A2: Expression in Pichia pastoris, Properties, and Application in Oil Degumming published in 2015 ) of Pichia pastoris competent, and construct a Pichia pastoris heat stress-related gene overexpression strain again. The single clone of the overexpressed strain was fermented in BMGY / BMMY medium. The...

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Abstract

The invention discloses a method for increasing the yield of pichia pastoris foreign protein, and belongs to the technical field of biological engineering. Through overexpression of the heat stress response related genes HSP78 and SSA3, activation of a heat stress response mechanism is promoted to help correct folding of intracellular proteins, so that the yield of pichia pastoris foreign proteins is increased. After the HSP78 and the SSA3 are over-expressed, the expression quantities of the green fluorescent protein, the lipase and the phospholipase of the strain are increased to different degrees, the expression quantity of the green fluorescent protein is increased by 50% at most, the expression quantity of the lipase is increased by 57% at most, and the expression quantity of the phospholipase is increased by 81% at most; therefore, the heat shock proteins HSP78 and SSA3 have better application value in expression of foreign proteins in pichia pastoris and improvement of the yield of the foreign proteins.

Description

technical field [0001] The invention relates to a method for increasing the output of exogenous protein of Pichia pastoris, in particular to a method for increasing the output of exogenous protein of Pichia pastoris by activating a heat stress response mechanism, and belongs to the technical field of bioengineering. Background technique [0002] Pichia pastoris is an important cell factory widely used in industrial production. Studies have reported that it can express more than 500 foreign proteins ranging from human endostatin to spider silk protein. Moreover, the Pichia pastoris expression system has many advantages in industrial application and laboratory level research, including: (1) The secretion of Pichia's own protein is small, and the secretion of foreign proteins in Pichia pastoris is as high as its total protein (2) Pichia pastoris can process and modify the translated protein as a eukaryotic expression system; (3) Pichia pastoris is easy to industrialize; (4) Pic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/67C12N15/31C07K14/435C12N9/18C12N9/20C12R1/84
CPCC07K14/39C12N15/815C12N15/67C07K14/43595C12N9/20C12N9/18C12Y301/01003C12Y301/01004
Inventor 喻晓蔚徐岩林乃馨
Owner JIANGNAN UNIV
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