NDLI: Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris

Content Provider	Springer Nature Link
Author	Damasce, Leonardo M. Anderson, Kyle A. Ritter, Gerd Cregg, James M. Old, Lloyd J. Batt, Carl A.
Copyright Year	2006
Abstract	In Pichia pastoris, secretion of the A33 single-chain antibody fragment (A33scFv) was shown to reach levels of approximately 4 g l−1 in fermentor cultures. In this study, we investigated whether manipulating chaperone and foldase levels in P. pastoris could further increase secretion of A33scFv. Cells were engineered to cooverexpress immunoglobulin binding protein (BiP) and/or protein disulfide isomerase (PDI) with A33scFv during growth in methanol as the sole carbon and energy source. Cooverexpression of BiP resulted in increased secretion levels of A33scFv by approximately threefold. In contrast, cooverexpression of PDI had no apparent effect on secretion of A33scFv. In cells cooverexpressing BiP and PDI, A33scFv secretion did not increase and protein levels remained the same as the control strain. We believe that secretion of A33scFv is increased by cooverexpression of BiP as a result of an increase in folding capacity inside the endoplasmic reticulum (ER). In addition, lack of increased single-chain secretion when PDI is coexpressed was unexpected due to the presence of disulfide bonds in A33scFv. We also show that during PDI cooverexpression with the single-chain there is a sixfold increase in BiP levels, indicating that the former is possibly inducing an unfolded protein response due to excess chaperone and recombinant protein in the ER.
Starting Page	381
Ending Page	389
Page Count	9
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	74
Issue Number	2
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2006-10-19
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Pichia pastoris scFv Chaperone Recombinant protein A33 Microbial Genetics and Genomics Microbiology Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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