A method for increasing the expression level of exogenous protein secreted by 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, so as to achieve the effect of increasing expression, improving correct folding, and promoting wide application
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Embodiment 1p
[0029] Embodiment 1 pGAPZA and pGAPZB plasmid construction
[0030] Plasmid pGAPZαA (purchased from Invitrogen Company) ( figure 1 ) with an α-factor signal peptide, suitable for secreting and expressing foreign proteins. When transforming Pichia pastoris hosts, first pass the AvrII restriction site and then linearize with AvrII. The linearized fragments can be better integrated into the host genome superior. The regulatory factors HAC1, ERO1, and BIP need to be expressed in the cell, 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 the plasmid pGAPZA, the restriction site AvrII was mutated to construct a new plasmid pGAPZB.
[0031] The specific construction process is as follows:
[0032] 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 reaction, so as to remove the inte...
Embodiment 2
[0042] Embodiment 2HAC1, ERO1, BIP gene amplification
[0043] The nucleotide sequences of HAC1, ERO1, and BIP genes were obtained from the KEGG database, which are respectively SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. These three genes were amplified using the following primers.
[0044] Primer 7 (HAC1-F): ATGCCCGTAGATTCTTCTC
[0045]Primer 8 (HAC1-R): TCACCTGATCGCTATGCAT
[0046] Primer 9 (ERO1-F): ATGAGGATAGTAAGGAGCG
[0047] Primer 10 (ERO1-R): TTACAAGTCTACTCTATAT
[0048] Primer 11 (BIP-F): ATGCTGTCGTTAAAACCAT
[0049] Primer 12 (BIP-R): CTACAACTCATCATGATCA
[0050] Using the Pichia pastoris GS115 genome as a template, primers 7 and 8 amplified to obtain the HAC1 gene with a size of 996 bp; primers 9 and 10 amplified to obtain the ERO1 gene with a size of 1584 bp; primers 11 and 12 amplified to obtain the BIP gene with a size of 2037 bp ; The three fragments were connected to the T vector and then sequenced, and the sequence was consistent with the sequence report...
Embodiment 3
[0051] Embodiment 3 pGAPHE, pGAPHEB plasmid construction
[0052] EcoR I and Not I restriction sites were introduced at both ends of the HAC1, ERO1 and BIP gene fragments by PCR. After double 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 ).
[0053] After the pGAPZB-ERO1 plasmid was digested with Bgl II and BamH I, the expression cassette containing the promoter, target gene and terminator was recovered from the gel, and the size was 2486bp. After the pGAPZA-HAC1 plasmid was digested with BamH I, it was connected with the expression cassette, transformed into DH5α strain, and positive transformants were picked. Since the restriction sites of Bgl II and BamHI are the same tail enzymes and have the same cohesive ends, two positive and reverse plasmids will be formed during ligation, which can be verified by extracting the plasmid a...
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