Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding.
The production of recombinant proteins is a critical technology for biotechnology and biomedical research. Heterologous expression of secreted proteins can saturate the cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. The overexpression of chaperone binding protein (BiP) and disulfide bond isomerase (PDI) in Saccaromyces cerevisiae can effectively increase protein production levels of single-chain antibody (scFv) 4-4-20. These studies show that overexpression of BiP did not reduce the UPR activated by heterologous protein expression; however, overexpression of PDI or co-overexpression of BiP and PDI could reduce the UPR. We observed that co-overexpression of BiP and PDI led to the greatest secretion of scFv from the cell, but BiP and PDI appear to interact with the newly synthesized scFv at different stages in the folding process, as determined by pulse-chase analysis. We propose that BiP acts primarily to facilitate translocation and retain unfolded or partially folded scFv, and PDI actively folds the scFv through its functions as a catalyst, and/or an isomerase, of disulfide bonds. Free BiP is released when scFv is folded, stabilizing Ire1p, and leading to the reduced UPR.
Xu
Ping
P
259 Colburn Laboratory, Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA.
Raden
David
D
Doyle
Francis J
FJ
Robinson
Anne Skaja
AS
D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000235:genetics*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D000911:Antibodies, Monoclonal; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; D019704:Protein Disulfide-Isomerases