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 (Delta N85 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 AN85 c-Src from recombinant baculovirus-infected insect cells. Q-Sepharose anion-exchange and amino-phenyl-ATP affinity chromatography were used to isolate 5mg of > 98% pure Delta N85 c-Src from 900mg of total soluble protein. The specific activity of Delta N85 c-Src (20 U mg(-1)) was found to be >= 5-fold greater than previously reported values. A lag in the autophosphorylation kinetics of AN85 c-Src was observed, and the reaction occurred with observed first-order rate constants k(1) = 0.20 +/- 0.01 min(-1) and k(2) = 0.38 +/- 0.01 min(-1) under the experimental conditions used. Steady-state kinetic analysis of peptide phosphorylation by Delta N85 c-Sre gave K Values of 99 +/- 23 mu M and 190 +/- 130 mu M for the peptide and ATP substrates, respectively, and a value of k(cat) = 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 AN85 c-Src required for rigorous structure-function and inhibition studies that rely on a pre-steady-state kinetic approach. (c) 2005 Elsevier Inc. All rights reserved.