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 subcloned into a plasmid vector expressing glutathione-S-transferase (GST). This new vector expresses a fusion protein in Escherichia coli composed 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-linked agarose. GST-kin (M(r) 57,000) is a phosphoprotein as judged by 32P autoradiography, consistent with the known autophosphorylation site within the kinase core [Weinmaster et al. (1984) Cell, 37, 559-568]. Cleavage of the fusion protein with thrombin and purification on phosphocellulose resin yielded the pure kinase domain (M(r) 33,000). The activity of the kinase domain is indistinguishable from that of GST-kin using the peptide substrate EEEIYEEIE, indicating that N-terminal fusion has no effect on the kinase domain. GST-kin phosphorylates a second peptide, EAEIYEAIE, with improved catalytic efficiency. Initial velocity data are consistent with a random bireactant mechanism with no substrate synergism observed in the ternary complex. Steady-state kinetic analyses reveal that this peptide is phosphorylated, with a kcat of 3.6 s-1, a Kpeptide of 500 microM and a KATP of 250 microM. The expression, purification and preliminary kinetic analysis of the kinase domain of v-fps provide the first step in the application of structure-function studies for this oncoprotein.
Gish
G
G
Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada.
McGlone
M L
ML
Pawson
T
T
Adams
J A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D015681:Fusion Proteins, gag-onc / Q000235:genetics* / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004274:DNA, Recombinant; D015681:Fusion Proteins, gag-onc; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; C068415:v-fps oncogene protein, Fujinami sarcoma virus