title: Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris.
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.
authors.affiliation: Department of Microbiology, Cornell University, Ithaca, NY 14853, USA. lmd42@cornell.edu.
authors.forename: Leonardo M
authors.initials: LM
authors.lastname: Damasceno
authors.forename: Kyle A
authors.initials: KA
authors.lastname: Anderson
authors.forename: Gerd
authors.initials: G
authors.lastname: Ritter
authors.forename: James M
authors.initials: JM
authors.lastname: Cregg
authors.forename: Lloyd J
authors.initials: LJ
authors.lastname: Old
authors.forename: Carl A
authors.initials: CA
authors.lastname: Batt
mesh_terms: D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods; D000091342:Endoplasmic Reticulum Chaperone BiP; D005818:Genetic Engineering / Q000379:methods; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D007128:Immunoglobulin Fragments; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D019704:Protein Disulfide-Isomerases