RESUMO
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.
Assuntos
Antígeno Carcinoembrionário/biossíntese , Pichia/genética , Sequência de Bases , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/isolamento & purificação , Cromatografia de Afinidade , Clonagem Molecular , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Fermentação , Glicosilação , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Transformação GenéticaRESUMO
Molecular farming in plants can be achieved by stable or transient expression of a recombinant protein. Transient expression of recombinant proteins in plants can rapidly provide large amounts of the proteins for detailed characterization. It is fast, flexible and can be carried out at field scale using viral vectors, but it lacks the increases in production volume that can be achieved easily with stable transgenic crops. This review article focuses on discussing the applications of transient expression using viral vectors, biolistic methods or agroinfiltration.