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 (DeltaN85 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 DeltaN85 c-Src from recombinant baculovirus-infected insect cells. Q-Sepharose anion-exchange and aminophenyl-ATP affinity chromatography were used to isolate 5mg of >98% pure DeltaN85 c-Src from 900 mg of total soluble protein. The specific activity of DeltaN85 c-Src (20 U mg(-1)) was found to be >or = 5-fold greater than previously reported values. A lag in the autophosphorylation kinetics of DeltaN85 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 DeltaN85 c-Src gave Km values of 99+/-23 microM and 190+/-30 microM for the peptide and ATP substrates, respectively, and a value of k(cat)=17+/-2s(-1). Overall, we present a dramatically improved purification strategy that represents a simplified, relatively rapid protocol for the isolation of milligram quantities of DeltaN85 c-Src required for rigorous structure-function and inhibition studies that rely on a pre-steady-state kinetic approach.
Jeansonne
Duane P
DP
Department of Biochemistry, Tulane University Health Sciences Center, 1430 Tulane Avenue SL 43, New Orleans, LA 70112, USA.
Bordes
Tammy J
TJ
Bennett
Cecily A
CA
Kothandaraman
Geetha
G
Bush
John G
JG
Vaccaro
Joseph A
JA
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D017384:Sequence Deletion; D018411:Spodoptera / Q000166:cytology / Q000235:genetics; D019061:src-Family Kinases
D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human