Development and characterization of a panel of monoclonal antibodies against the catalytic domain of the human fes proto-oncogene product

• Published in: Molecular biology reports Vol. 16; no. 1; pp. 17 - 25
• Main Authors: VAN BOKHOVEN, A, VAN DUIJNHOVEN, H. L. P, JÜCKER, M, ROEBROEK, A. J. M, VAN DE VEN, W. J. M
• Format: Journal Article
• Language: English
• Published: Dordrecht Kluwer 01-02-1992
• Subjects: Adenosine Triphosphate - metabolism; Animals; Antibodies, immunoglobulins; Antibodies, Monoclonal - immunology; Binding Sites; Biological and medical sciences; Blotting, Western; Cell Line; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Molecular immunology; Monoclonal antibodies; Protein-Tyrosine Kinases; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - immunology; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-fes; Proto-Oncogenes; Recombinant Fusion Proteins - immunology; Human; Catalytic site; Monoclonal antibody; Gene product; C-Onc gene; Protooncogene
• ISSN: 0301-4851; 1573-4978
• DOI: 10.1007/BF00788749

In developing monoclonal antibodies (Moabs) against the human fes proto-oncogene product, recombinant DNA technology was used to target reactivity of the Moabs towards the catalytic domain of it. Therefore, sequences of human fes exons 15-19 encoding amino acid residues 612 to 822 which harbor the catalytic domain except the presumed ATP-binding region, were fused in phase to the bacterial trp E gene which encodes anthranilate synthase. After partial purification of it, the bacterially produced hybrid product of this trp E-delta fes fusion gene was used as immunogen. A series of twelve mouse Moabs was obtained which recognized the human p92fes protein and the viral oncogene product p85gag-fes encoded by the Snyder-Theilen strain of feline sarcoma virus. Reactivity appeared to be directed towards the catalytic domain of the human fes proto-oncogene product. This was demonstrated by in vitro transcription and translation experiments using human fes coding sequences from exons 16-19. Upon testing their functional activity in divers immunological techniques, the whole panel of Moabs appeared to be useful in immunoprecipitation, Western blot and immunohistochemical analysis. Immunocytochemical analysis indicated that p85gag-fes is predominantly a cytoplasmic protein.