Characterization of the Kinase Domain of the Ephrin-B3 Receptor Tyrosine Kinase Using a Scintillation Proximity Assay

Abstract

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 ∼0.4 μM. The apparent K _m for ATP was determined to be ∼3 μM. 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 h at room temperature. Moreover, the tryrosine kinase inhibitor, PP2, produced an IC₅₀ of roughly 0.8 μM. In addition, the enzyme tolerated the solvent DMSO and was stable to multiple freeze/thaw cycles. Stability of the enzyme at 4°C storage was seen out to 6 h with an ∼50% 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 ∼24 and a z value of 0.72. Collectively, these results showed the ability to configure a robust HTS for a truncated recombinantly expressed family member of the Ephrin tyrosine kinases.