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Applied and Environmental Microbiology, September 2005, p. 5332-5340, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5332-5340.2005

Overexpression of an Anti-CD3 Immunotoxin Increases Expression and Secretion of Molecular Chaperone BiP/Kar2p by Pichia pastoris

Yuan Yi Liu,^* Jung Hee Woo, and David M. Neville Jr.

Section on Biophysical Chemistry, Laboratory of Molecular Biology, National Institute of Mental Health, Building 10, Rm. 3D46, 10 Center Drive, Bethesda, Maryland 20892-1216

Received 7 February 2005/ Accepted 1 April 2005

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 -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.

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* Corresponding author. Mailing address: National Institute of Mental Health, Bldg. 10, Rm. 3D46, 10 Center Drive, Bethesda, MD 20892-1216. Phone: (301) 402-0651. Fax: (301) 480-0446. E-mail: yyliu{at}mail.nih.gov.

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Applied and Environmental Microbiology, September 2005, p. 5332-5340, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5332-5340.2005

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