Overexpression of an anti-CD3 immunotoxin increases expression and secretion of molecular chaperone BiP/Kar2p by Pichia pastoris.
We previously reported that the secretory capacity of Pichia pastoris is limited with respect to the secretion of a 96.5-kDa bivalent anti-CD3 immunotoxin; double-copy expression generated more translation products than single-copy expression but did not increase the secretion of the immunotoxin. In Saccharomyces cerevisiae heterologous protein secretion has been reported to increase the expression of molecular chaperones, most prominently BiP/Kar2p. We therefore investigated the relationships between immunotoxin secretion and Kar2p expression in P. pastoris. We found that expression of the immunotoxin in P. pastoris increased the expression of Kar2p to levels that surpassed the retrieval capacity of the cell, leading to secretion of Kar2p into the medium. The level of Kar2p secretion was correlated with the copy number of the immunotoxin gene. Intracellular Kar2p was found to bind exclusively to the unprocessed immunotoxin containing the prosequence of alpha-factor in the endoplasmic reticulum. These results show that Kar2p is intimately involved in immunotoxin secretion in P. pastoris. The limited capacity of P. pastoris to retain a sufficiently high level of intracellular Kar2p may be a factor restricting the production of the immunotoxin.
Liu
Yuan Yi
YY
National Institute of Mental Health, Bldg. 10, Rm. 3D46, 10 Center Drive, Bethesda, MD 20892-1216. USA. yyliu@mail.nih.gov
Woo
Jung Hee
JH
Neville
David M
DM
D001709:Biotechnology / Q000379:methods; D017252:CD3 Complex / Q000276:immunology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D015966:Gene Expression Regulation, Fungal; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D000922:Immunotoxins / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D017252:CD3 Complex; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D000922:Immunotoxins; D018832:Molecular Chaperones; D011994:Recombinant Proteins