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Protein disulfide isomerase, but not binding protein, overexpression enhances secretion of a non-disulfide-bonded protein in yeast.

Abstract：

:In eukaryotes, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). Many heterologous proteins are retained in the ER due to suboptimal folding conditions. We previously reported that heterologous secretion of Pyrococcus furiosus beta-glucosidase in Saccharomyces cerevisiae resulted in the accumulation of a large fraction of inactive beta-glucosidase in the ER. In this work, we determine the effect of introducing additional genes of ER-resident yeast proteins, Kar2p (binding protein [BiP]) and protein disulfide isomerase (PDI), on relieving this bottleneck. Single-copy expression of BiP and PDI worked synergistically to improve secretion by reverse similar 60%. In an effort to optimize BiP and PDI interactions, we created a library of beta-glucosidase expression strains that incorporated four combinations of constitutively or inducibly-expressed BiP and PDI genes integrated to random gene copynumbers in the yeast chromosome. Approximately 15% of the transformants screened had secretion level improvements higher than that seen with single BiP/PDI gene overexpression, and the highest secreting strain had threefold higher beta-glucosidase levels than the control. Nineteen of the improved strains were re-examined for beta-glucosidase secretion as well as BiP and PDI levels. Within the improved transformants BiP and PDI levels ranged sevenfold and tenfold over the control, respectively. Interestingly, increasing BiP levels decreased beta-glucosidase secretion, whereas increasing PDI levels increased beta-glucosidase secretion. The action of PDI was unexpected because beta-glucosidase is not a disulfide-bonded protein. We suggest that PDI may be acting in a chaperone-like capacity or possibly creating mixed disulfides with the beta-glucosidase's lone cysteine residue during the folding and assembly process.

journal_name

Biotechnol Bioeng

journal_title

Biotechnology and bioengineering

authors

Smith JD,Tang BC,Robinson AS

doi

10.1002/bit.10853

subject

Has Abstract

pub_date

2004-02-05 00:00:00

pages

340-50

issue

eissn

0006-3592

issn

1097-0290

journal_volume

pub_type

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Protein disulfide isomerase, but not binding protein, overexpression enhances secretion of a non-disulfide-bonded protein in yeast.