Method for improving expression amount of secretory foreign protein in pichia pastoris

A Pichia pastoris and exogenous protein technology, applied in the field of genetic engineering, can solve the problems of low secretion and expression of exogenous proteins and large differences in expression, and achieve the effect of increasing expression, improving correct folding, and promoting wide application

Active Publication Date: 2014-11-19
QINGDAO VLAND BIOTECH GRP
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, with the expression of a large number of different foreign proteins in Pichia pastoris, it was found that the expression of different foreign proteins in Pichia pastoris was quite different
Existing studies mainly focus on the relationship between the secretion and expression of exogenous proteins and factors such as gene copy number and messenger RNA content. However, recent studies have found that the low secretion and expression of exogenous proteins is often due to the caused by the aggregation of

Method used

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  • Method for improving expression amount of secretory foreign protein in pichia pastoris
  • Method for improving expression amount of secretory foreign protein in pichia pastoris
  • Method for improving expression amount of secretory foreign protein in pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1p

[0028] Example 1 Construction of pGAPZA and pGAPZB plasmids

[0029] Plasmid pGAPZαA (purchased from Invitrogen) ( figure 1 ) with α-factor signal peptide, suitable for secreting and expressing exogenous protein, when transforming Pichia pastoris host, it is firstly digested with AvrII enzyme through the AvrII restriction site and then linearized, and the linearized fragment can be better integrated into the host genome superior. The regulatory factors HAC1, ERO1, and BIP need to be expressed intracellularly, so it is necessary to transform the plasmid pGAPZαA, remove the signal peptide, and construct a new plasmid pGAPZA ( figure 2 ), and on the basis of plasmid pGAPZA, the restriction enzyme site AvrII was mutated to construct a new plasmid pGAPZB.

[0030] The specific construction process is as follows:

[0031] The gene fragments of promoter pGAP and terminator AOX1TT were cloned by PCR reaction, and then the two fragments were fused together by overlapping PCR reacti...

Embodiment 2

[0041] Example 2 HAC1, ERO1, BIP gene amplification

[0042] The nucleotide sequences of HAC1, ERO1, and BIP genes were obtained from the KEGG database, which are SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. These three genes were amplified using the primers described below.

[0043] Primer 7 (HAC1-F): ATGCCCGTAGATTCTTCTC

[0044]Primer 8 (HAC1-R): TCACCTGATCGCTATGCAT

[0045] Primer 9 (ERO1-F): ATGAGGATAGTAAGGAGCG

[0046] Primer 10 (ERO1-R): TTACAAGTCTACTCTATAT

[0047] Primer 11 (BIP-F): ATGCTGTCGTTAAAACCAT

[0048] Primer 12 (BIP-R): CTACAACTCATCATGATCA

[0049] Using the genome of Pichia pastoris GS115 as the template, primers 7 and 8 were amplified to obtain the HAC1 gene with a size of 996bp; primers 9 and 10 were amplified to obtain the ERO1 gene with a size of 1584bp; primers 11 and 12 were amplified to obtain the BIP gene with a size of 2037bp ; The 3 fragments were connected to T vector and sequenced, and the sequence was consistent with the seq...

Embodiment 3

[0050] Example 3 Construction of pGAPHE and pGAPHEB plasmids

[0051] EcoR I and Not I restriction sites were introduced at both ends of the HAC1, ERO1 and BIP gene fragments by PCR. After double restriction digestion, HAC1 was connected to the pGAPZA plasmid, and ERO1 and BIP were connected to the pGAPZB plasmid to construct pGAPZA-HAC1 and pGAPZB-ERO1 respectively. , pGAPZB-BIP plasmid ( image 3 ).

[0052] After the pGAPZB-ERO1 plasmid was double digested with Bgl II and BamH I, the expression cassette containing the promoter, the target gene and the terminator was recovered by gel, and the size was 2486bp. After the pGAPZA-HAC1 plasmid was digested with BamH I, it was ligated with the expression cassette, transformed into the DH5α strain, and the positive transformants were picked. Since the digestion sites of Bgl II and BamHI are homo-caudal enzymes and have the same sticky ends, two plasmids, forward and reverse, will be formed when ligating, which can be verified by ...

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Abstract

The invention aims to provide a method for improving the expression amount of secretory foreign protein in pichia pastoris. Byintracellular co-expression of double genes of HAC1 and ERO1 or three genes of HAC1, ERO1 and BIP in pichia pastoris, the expression amount of secretory foreign protein in pichia pastoris is effectively improved. According to the invention, double genes of HAC1 and ERO1 or three genes of HAC1, ERO1 and BIP are transformed into pichia pastoris secreting and expressing exogenous xylanase to obtain recombinant pichia pastoris strains, thereby significantly increasing the expression amount of the exogenous xylanase. During co-expression of double genes of HAC1 and ERO1 in pichia pastoris, the secretion expression amount of exogenous xylanase is generally increased by 15%-25% compared with the initial level; and during the co-expression of three genes HAC1, ERO1 and BIP, the secretion expression amount of xylanase is increased by 45%-57% compared with the initial level.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for increasing the expression of secreted exogenous protein of Pichia pastoris. technical background [0002] Pichia pastoris, as an important host for the expression of exogenous proteins, has many advantages: simple culture method, genetically stable strain, high protein expression, and the expressed protein can be correctly folded and post-translationally modified. However, with the expression of a large number of different exogenous proteins in Pichia pastoris, it was found that the expression levels of different exogenous proteins in Pichia pastoris were quite different. Existing studies have mainly focused on the relationship between the secretion and expression of exogenous proteins and factors such as gene copy number and messenger RNA content. caused by the aggregation. By adopting different promoters or strategies to increase gene copy number to e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12R1/84
Inventor 徐晓东王华明李冬冬王海
Owner QINGDAO VLAND BIOTECH GRP
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