Development and characterization of a panel of monoclonal antibodies against the catalytic domain of the human fes proto-oncogene product.
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.
van Bokhoven
A
A
Department of Biochemistry, University of Nijmegen, The Netherlands.
van Duijnhoven
H L
HL
Jücker
M
M
Roebroek
A J
AJ
van de Ven
W J
WJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D011505:Protein-Tyrosine Kinases; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D051578:Proto-Oncogene Proteins c-fes; D011519:Proto-Oncogenes; D011993:Recombinant Fusion Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D051578:Proto-Oncogene Proteins c-fes