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Biotechnology and Applied Biochemistry (1999) 30, (267–275) (Printed in Great Britain)
Production of carcinoembryonic antigen (CEA) N-A3 domain in Pichia pastoris by fermentation
Stephan Hellwig*, Frederic Robin*, Jürgen Drossard*, Nicole P. G. Raven*, Carmen Vaquero-Martin*, John E. Shively† and Rainer Fischer*‡1
*Institut für Biologie I (Botanik/Molekulargenetik), RWTH Aachen, D-52074 Aachen, Germany, †Department of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, U.S.A., and ‡Fraunhofer Department for Molecular Biotechnology, IUCT, Grafschaft, Auf dem Aberg 1, D-57392 Schmallenberg, Germany
Abbreviations used: YPD medium, yeast extract/peptone/dextrose medium; PNGase F, peptide N-glycosidase F; BMGY, buffered complex medium containing glycerol as the sole carbon source; BMMY, buffered complex medium containing methanol as the sole carbon source; CEA, carcinoembryonic antigen; GAM-AP, alkaline phosphatase-conjugated goat anti-mouse Fc-specific polyclonal secondary antibody; NBT-BCIP, Nitro Blue Tetrazolium chloride/5-bromo-4-chloroindol-3-yl phosphate; NTA, nitrilotriacetic acid; IMAC, immobilized metal-affinity chromatography. 1 To whom correspondence should be addressed, at the Institut für Biologie I (Botanik/Molekulargenetik). 
Carcinoembryonic antigen (CEA) is a 180-kDa glycoprotein found on the surface of normal colon and malignant human adenocarcinomas. Recently, a fusion protein containing two of the seven Ig-like domains present in CEA (N and A3) has been constructed and expressed in Pichia pastoris [You, Hefta, Yazaki, Wu and Shively (1998) Anticancer Res. 18, 3193–3201]. Here, we report the generation and selection of a multi-copy clone expressing this fusion protein, the optimization of the shake-flask expression protocol and the upscaled production of CEA N-A3 using fermentation technology. P. pastoris transformants secreting the CEA N-A3 domain were generated by electrotransformation of the GS115 host strain with the pPIC9K vector containing the CEA N-A3 cDNA [You, Hefta, Yazaki, Wu and Shively (1998) Anticancer Res. 18, 3193–3201] then screened for CEA N-A3 expression and G418 resistance. The recombinant CEA N-A3 domain was detected in the culture supernatant using the monoclonal anti-CEA antibody T84.66. Optimization of methanol-induction conditions resulted in a high-methanol shake-flask expression protocol yielding significantly increased CEA N-A3 levels. Fermentation and culture conditions were optimized for 5-l working-volume fermentations and CEA N-A3 was affinity purified using Ni-IDA (imino di-acetic acid) affinity chromatography from the clarified fermentation supernatant. Peptide N-glycosidase F treatment revealed that the recombinant protein was heavily glycosylated but expressed as a single polypeptide of 28 kDa with no evidence of proteolytic degradation. Our results demonstrate that functional CEA N-A3 domain can be produced in sufficient quantities in P. pastoris for structural analysis or diagnostic applications. To our knowledge, this article represents the first report on the production of a human tumour antigen through fermentation.
Received 14 July 1999; accepted 17 August 1999
Portland Press Ltd © 1999 |
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