Production of carcinoembryonic antigen (CEA) N-A3 domain in Pichia pastoris by fermentation

Carcinoembryonic antigen (CEA) is a 180-kDa glycoprotein found on the surfa ce of normal colon and malignant human adenocarcinomas, Recently, a fusion protein containing two of the seven Ig-like domains present in CEA (N and A 3) has been constructed and expressed in Pichia pastoris [You, Hefta, Yazak i, Wu and Shivery (1998) Anticancer Res, 18, 3193-3201], Here, we report th e generation and selection of a multicopy clone expressing this fusion prot ein, the optimization of the shake-flask expression protocol and the upscal ed production of CEA N-A3 using fermentation technology, P, pastoris transf ormants secreting the CEA H-A3 domain were generated by electrotransformati on of the GS115 host strain with the pPIC9K vector containing the CEA N-A3 cDNA [You, Hefta, Yazaki, Wu and Shivery (1998) Anticancer Res, 18, 3193-32 01] then screened for CEA N-A3 expression and G418 resistance. The recombin ant CEA N-A3 domain was detected in the culture supernatant using the monoc lonal anti-CEA antibody T84.66. Optimization of methanol-induction conditio ns resulted in a high-methanol shake-flask expression protocol yielding sig nificantly increased CEA N-A3 levels. Fermentation and culture conditions w ere optimized for 5-1 working volume fermentations and CEA N-A3 was affinit y 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 expresse d as a single polypeptide of 28 kDa with no evidence of proteolytic degrada tion. Our results demonstrate that functional CEA N-A3 domain can be produc ed in sufficient quantities in P. pastoris for structural analysis or diagn ostic applications, To our knowledge, this article represents the first rep ort on the production of a human tumour antigen through fermentation.