Construction of a cDNA for the human c-fes protooncogene protein-tyrosine kinase and its expression in a baculovirus system.
Previous studies have established that the 93-kDa protein-tyrosine kinase (PTK) encoded by the human c-fes protooncogene plays an active role in the induction of terminal myeloid differentiation. However, this enzyme is expressed at very low levels in myeloid cells, making isolation of sufficient quantities for detailed biochemical analysis difficult. To overcome this problem, we used the polymerase chain reaction to construct a full-length c-fes cDNA from overlapping 5' and 3' partial cDNA sequences. The c-fes cDNA was expressed at high levels in a baculovirus system, and the catalytically active recombinant c-fes gene product p93c-fes was partially purified by DEAE-Sepharose and tyrosine-agarose chromatography. Recombinant p93c-fes was indistinguishable from the native protein in terms of its apparent molecular weight following SDS-PAGE, catalytic activity, Km for poly(Glu,Tyr)4:1, antigenicity, and phosphopeptide pattern generated with Staphylococcus aureus protease.
Smithgall
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TE
Department of Pharmacology, Georgetown University Medical Center, Washington, D.C. 20007.
Goswami
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BB
Nagashfar
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Ahmad
S
S
Glazer
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RI
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics*; D051578:Proto-Oncogene Proteins c-fes; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D004247:DNA; D011505:Protein-Tyrosine Kinases; C496645:FES protein, human; D051578:Proto-Oncogene Proteins c-fes