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Pathology & Oncology Research

, 11:211 | Cite as

Effective production of carcinoembryonic antigen by conversion of the membrane-bound into a recombinant secretory protein by site-specific mutagenesis

  • Fakhraddin Naghibalhossaini
  • Abbas Pakdel
  • Abbas Ali Ghaderi
  • Mehdi Saberi Firoozi
Article

Abstract

Carcinoembryonic antigen (CEA), the most widely used human tumor marker, is a heavily glycosylated protein over-expressed by a wide range of tumors. It has been indicated that CEA might be a useful target for human anti-tumor immunotherapy. CEA assay for research as well as clinical trials demands a continuous source of CEA protein preparations. In a multi-purpose research program to provide a reliable source for large production of CEA, we converted the membrane-bound carcinoembryonic antigen into a secretory protein by site-specific mutagenesis. We made the secretory CEA protein by introducing a new stop codon at 99 bp upstream of the original stop codon in CEA cDNA by PCR-based mutagenesis. The glycosylation of recombinant CEA proteins, especially those destined for administration to human trials is crucially important. To produce CEA with the same glycosylation pattern and immunogenicity as the native CEA expressed by human tumorsin vivo, the truncated CEA cDNA which does not encode the last C-terminal 33-amino acid hydrophobic domain was transfected into HT29, a human colon carcinoma cell line by the calcium phosphate method. Stable transfectants were selected and pooled. CEA secretion from the cells was verified by analysis of the transfectant culture supernatant for CEA protein. As determined by ELISA, 16 μg/L of recombinant CEA was secreted per 106 transfectants within 48 hrs, an increase over 40 times relative to the untransfected cells. The size of the recombinant CEA secreted by HT29 transfectants in our experiment is identical to that of reference CEA secreted from tumors and is fully antigenic. It seems that the C-terminal truncation does not affect CEA glycosylation in HT29 cells. It is predicted that human cancer immunotherapy using recombinant CEA expressed in this system would be more effective than the commercial protein which is usually prepared from bacterial or other heterologous expression systems.

Key words

CEA GPI site-specific mutagenesis immunotherapy 

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Copyright information

© Arányi Lajos Foundation 2005

Authors and Affiliations

  • Fakhraddin Naghibalhossaini
    • 1
  • Abbas Pakdel
    • 1
  • Abbas Ali Ghaderi
    • 2
  • Mehdi Saberi Firoozi
    • 3
  1. 1.Department of Biochemistry, School of MedicineShiraz University of Medical SciencesShirazIran
  2. 2.Department of ImmunologyShiraz University of Medical SciencesIran
  3. 3.Gastroenterohepatology Research CenterShiraz University of Medical SciencesIran

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