Alteration of hybridoma viability and antibody secretion in transfectomas with inducible overexpression of protein disulfide isomerase.
Monoclonal antibody (mAb)-secreting transfectomas with dexamethasone inducible expression of the mammalian endoplasmic reticulum foldase and chaperone protein disulfide isomerase (PDI, ERp59) were generated from the murine 9.2.27 hybridoma in order to obtain in vivo evidence of whether alteration of the level of PDI, believed to be involved in immunoglobulin (Ig) assembly, results in alteration of mAb secretion kinetics. Using an RNase refolding assay, the specific activity of endogenous PDI in the 9.2.27 hybridoma was found to be constant during batch growth. An expression vector for glucocorticoid-inducible overexpression of PDI, pMMTVPDI, was constructed from pMAMneo using a rat PDI cDNA. Cell lysates of stable transfectomas contained 2-4-fold higher levels of PDI mRNA and increased levels of PDI protein, detected by immunoblotting, following induction with 0.1 microM dexamethasone. Monoclonal antibody secretion kinetics were evaluated in 12.5 mL shake flasks, a 100 mL spinner, and a 1 L aerated batch reactor. A transfectoma was found with altered mAb secretion kinetics during cell growth following dexamethasone induction of PDI overexpression. Specific mAb secretion rate was not significantly increased following dexamethasone induction; however, hybridoma viability was sustained longer during the stationary phase of cell growth and hence total antibody yield was increased in comparison to the parent 9.2.27 hybridoma.
Department of Biochemistry, University of Minnesota, St. Paul 55108-6106, USA.
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000502:physiology*; D000951:Antigens, Neoplasm / Q000276:immunology; D002417:Cattle; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004790:Enzyme Induction; D006825:Hybridomas / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D007074:Immunoglobulin G / Q000502:physiology; D007535:Isomerases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008545:Melanoma / Q000276:immunology; D051379:Mice; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D051381:Rats; D014162:Transfection
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D007074:Immunoglobulin G; D003907:Dexamethasone; D007535:Isomerases; D019704:Protein Disulfide-Isomerases