Purification and characterization of P47gag-crk expressed in insect cells.

Abstract

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.