In:
Protein & Peptide Letters, Bentham Science Publishers Ltd., Vol. 28, No. 12 ( 2021-12), p. 1434-1441
Abstract:
Pichia pastoris is one of the most popular eukaryotic hosts for producing
heterologous proteins, while increasing the secretion of target proteins is still a top priority for their application in industrial fields. Recently, the research effort to enhance protein production has focused
on up-regulating the unfolded protein response (UPR). Objective: We evaluated the effects of activated UPR via Hac1p co-expression with the promoter
AOX1 (PAOX1) or GAP (PGAP) on the expression of recombinant chitosanase (rCBS) in P. pastoris. Method: The DNA sequence encoding the chitosanase was chemically synthesized and cloned into
pPICZαA, and the resulting pPICZαA/rCBS was transformed into P. pastoris for expressing rCBS. The P. pastorisHAC1i cDNA was chemically synthesized and cloned into pPIC3.5K to give
pPIC3.5K/Hac1p. The HAC1i cDNA was cloned into PGAPZB and then inserted with the HIS4 gene from pAO815 to construct the vector PGAPZB/Hac1p/HIS4. For co-expression of Hac1p, the
two plasmids pPIC3.5K/Hac1p and PGAPZB/Hac1p/HIS4 were transformed into P. pastoris harboring the CBS gene. The rCBS was assessed based on chitosanase activity and analyzed by SDSPAGE.
The enhanced Kar2p was detected with western blotting to evaluate UPR. Results: Hac1p co-expression with PAOX1 enhanced rCBS secretion by 41% at 28°C. Although
the level of UPR resulting from Hac1p co-expression with PAOX1 was equivalent to that with PGAP in terms of the quantity of Kar2p (a hallmark of the UPR), substitution of PGAP for PAOX1
further increased rCBS production by 21%. The methanol-utilizing phenotype of P. pastoris did not affect rCBS secretion with or without co-expression of Hac1p. Finally, Hac1p co-expression
with PAOX1 or PGAP promoted rCBS secretion from 22 to 30°C and raised the optimum induction temperature. Conclusion: The study indicated that Hac1p co-expression with PAOX1 or PGAP is an effective
strategy to trigger UPR of P. pastoris and a feasible means for improving the production of rCBS therein.
Type of Medium:
Online Resource
ISSN:
0929-8665
DOI:
10.2174/0929866528666211105111155
Language:
English
Publisher:
Bentham Science Publishers Ltd.
Publication Date:
2021
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