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.

A Yeast Platform for the Production of Single-Chain Antibody-Green Fluorescent Protein Fusions

“…The endoplasmic reticulum has often been recognized as a bottleneck for foreign-protein secretion in yeast (31,32). Manipulation of levels of ER chaperones and foldases improves secretion levels for many yeast foreign proteins, including scFv (9,28,31,33), but overexpression of ER chaperones and foldases does not always help foreign-protein secretion (30,33). Similarly, BiP and PDI did not significantly affect the secretion of fusion proteins in this study, and large amounts of protein were retained intracellularly in a terminal bottleneck.…”

Modulation of Chaperone Gene Expression in Mutagenized Saccharomyces cerevisiae Strains Developed for Recombinant Human Albumin Production Results in Increased Production of Multiple Heterologous Proteins

“…Data were restricted to combined overexpression of SIL1, LHS1, and JEM1 in the strain DYB7. Many studies in this field have shown that the beneficial effects of chaperone overexpression are highly substrate dependent (13,43). In this case all three proteins, with distinct characteristics (Table 5), showed increased production levels from shake-flask culture.…”

Effects of overexpression of X‐box binding protein 1 on recombinant protein production in Chinese hamster ovary and NS0 myeloma cells

“…While such a strategy increases heterologous protein production in yeast (Shusta et al, 1998;Smith et al, 2004) and insect cells (Ailor and Betenbaugh, 1998;Hsu and Betenbaugh, 1997), it has not consistently achieved the same effects in mammalian cells (Borth et al, 2005;Davis et al, 2000;Kitchin and Flickinger, 1995). Surprisingly, the reduction of the level of endogenous BiP, rather than its overexpression, was found to improve secretion of tissue plasminogen activator in CHO cells (Dorner et al, 1988).…”