A rapid ATP affinity-based purification for the human non-receptor tyrosine kinase c-Src

The non-receptor tyrosine kinase c-Src plays a central role in a variety of cell signaling pathways that regulate cell growth, differentiation, apoptosis, and other important cellular processes. An 85-amino acid N-terminal deletion construct of c-Src (ΔN85 c-Src) has been structurally characterized and used extensively in biochemical and biophysical studies. In this report, we have established a relatively rapid, simplified purification of ΔN85 c-Src from recombinant baculovirus-infected insect cells. Q-Sepharose anion-exchange and aminophenyl-ATP affinity chromatography were used to isolate 5 mg of >98% pure ΔN85 c-Src from 900 mg of total soluble protein. The specific activity of ΔN85 c-Src (20 U mg−1) was found to be ⩾5-fold greater than previously reported values. A lag in the autophosphorylation kinetics of ΔN85 c-Src was observed, and the reaction occurred with observed first-order rate constants k1 = 0.20 ± 0.01 min−1 and k2 = 0.38 ± 0.01 min−1 under the experimental conditions used. Steady-state kinetic analysis of peptide phosphorylation by ΔN85 c-Src gave Km values of 99 ± 23 μM and 190 ± 30 μM for the peptide and ATP substrates, respectively, and a value of kcat = 17 ± 2 s−1. Overall, we present a dramatically improved purification strategy that represents a simplified, relatively rapid protocol for the isolation of milligram quantities of ΔN85 c-Src required for rigorous structure–function and inhibition studies that rely on a pre-steady-state kinetic approach.