BACTERIAL EXPRESSION, PURIFICATION AND PRELIMINARY KINETIC DESCRIPTION OF THE KINASE DOMAIN OF V-FPS

The gene coding for the tyrosine protein kinase domain of v-fps was su bcloned into a plasmid vector expressing glutathione-S-transferase (GS T). This new vector expresses a fusion protein in Escherichia coil com posed of the kinase domain linked with GST at the N-terminus (GST-kin) . A portion of the total expressed protein was soluble upon cell lysis and was purified by affinity chromatography using glutathione cross-l inked agarose, GST-kin (M(r) 57 000) is a phosphoprotein as judged by P-32 autoradiography, consistent with the known autophosphorylation si te within the kinase core [Weinmaster et al, (1984) Cell, 37, 559-568] . Cleavage of the fusion protein with thrombin and purification on pho sphocellulose resin yielded the pure kinase domain (M(r) 33 000). The activity of the kinase domain is indistinguishable from that of GST-ki n using the peptide substrate EEEIYEEIE, indicating that N-terminal fu sion has no effect on the kinase domain, GST-kin phosphorylates a seco nd peptide, EAEIYEAIE, with improved catalytic efficiency. Initial vel ocity data are consistent with a random bireactant mechanism with no s ubstrate synergism observed in the ternary complex. Steady-state kinet ic analyses reveal that this peptide is phosphorylated, with a k(cat) of 3.6 s(-1), a K-peptide of 500 mu M and a K-ATP of 250 mu M. The exp ression, purification and preliminary kinetic analysis of the kinase d omain of v-fps provide the first step in the application of structure- function studies for this oncoprotein.