Characterization of the kinase domain of the ephrin-B3 receptor tyrosine kinase using a scintillation proximity assay

We have characterized a recombinantly expressed N-terminally tagged GST fusion of the tyrosine kinase domain of human EphB3. The EphB3 kinase domain was shown to phosphorylate a group of synthetic tyrosine-containing peptides derived from a proprietary biotinylated kinase-biased peptide substrate library. In addition, the enzyme activity was stimulated by the divalent cation, manganese, and inhibited by addition of magnesium. The most active tyrosine-containing peptide, a biotinylated 49-mer, displayed saturation kinetics with an apparent K-m of similar to0.4 muM. The apparent K-m for ATP was determined to be similar to3 muM. The kinetics of the reaction was linear from concentrations of enzyme of 0.5 to 2 nM, and at or below the K-m concentrations of the two substrates for at least 2 It at room temperature. Moreover, the tryrosine kinase inhibitor, PP2, produced an IC50 of roughly 0.8 muM. In addition, the enzyme tolerated the solvent DMSO and was stable to multiple freeze/thaw cycles. Stability of the enzyme at 4degreesC storage was seen out to 6 It with an similar to50% reduction of activity by 24 h. Formatting the assay in a 384-well microtiter plate produced good uniformity of signal at 100% inhibition, 50% inhibition, and no inhibition. The coefficient of variance was at or below 10% with a signal-to-background ratio of similar to24 and a z value of 0.72. Collectively, these results showed the ability to configure a robust FITS for a truncated recombinantly expressed family member of the Ephrin tyrosine kinases.