Purification and characterization of P47gag-crk expressed in insect cells.
The crk oncogene product, P47gag-crk, was expressed and purified using a baculovirus expression system. Approximately 2-10 mg of P47gag-crk was produced in 10(9) insect cells infected with a recombinant baculovirus. Partially purified P47gag-crk was obtained by precipitation in a low salt buffer and gel filtration. A better purification of P47gag-crk was achieved by immunoaffinity chromatography, resulting in a single band by Coomassie Blue staining. The insect cells expressing P47gag-crk showed an increase in protein-phosphotyrosine content, which is a characteristic feature of crk-transformed cells. Moreover, like P47gag-crk produced in chicken or rat cells, P47gag-crk produced in insect cells associated in vitro with a tyrosine kinase and its substrates from Crk-3Y1 cells. Peptide mapping of P47gag-crk expressed in insect, rat, and chicken cells showed that similar sites were phosphorylated in these proteins. These data suggest that P47gag-crk expressed in insect cells is functional and will be useful for the further analysis of this protein.
Matsuda
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Rockefeller University, New York, New York 10021.
Marshall
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CP
Hanafusa
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D000818:Animals; D000911:Antibodies, Monoclonal; D001358:Avian Sarcoma Viruses / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007304:Insect Viruses / Q000235:genetics; D007313:Insecta; D050736:Oncogene Protein v-crk; D009857:Oncogenes; D010449:Peptide Mapping; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000302:isolation & purification; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification; D012191:Retroviridae Proteins / Q000235:genetics* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D050736:Oncogene Protein v-crk; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D012191:Retroviridae Proteins; C060731:p47 (gag-crk) protein, Avian sarcoma virus; D011505:Protein-Tyrosine Kinases