Recombinant pp60c-src from baculovirus-infected insect cells: purification and characterization.
A simple and effective method has been developed to purify the recombinant protein tyrosine kinase pp60c-src from a baculovirus-insect cell expression system. The procedure includes affinity chromatography and HPLC. Milligram quantities of protein have been isolated with an activity of 3.9 mumol/min/mg protein using the substrate poly E4Y. This specific activity is many times higher than any published protocol. The enzyme is stable for months when stored in buffered 10% glycerol at -70 degrees C. This purification technique is compared to the immuno-affinity technique which is widely used for this enzyme. Enzyme kinetics were characterized with respect to substrate specificity, the effect of temperature, ionic strength, pH, and Mg+2 versus Mn+2 ions. Similar to the enzyme expressed in human cells, the recombinant enzyme demonstrated a higher Vmax and substrate specificity for poly E4Y over 5V-Agt-II. An activation energy of 14.2 kcal/mol was determined. Inhibition by increasing ionic strength is mostly due to an increase in Km for the poly E4Y substrate and hence was substrate dependent. The Km(ATP) was pH dependent while the Km(poly E4Y) was pH independent. For the phosphorylation of poly E4Y, free Mg+2 was stimulatory while Mn+2 was inhibitory. In contrast, Mn+2 stimulated the phosphorylation of 5V-Agt-II.
Budde
R J
RJ
Department of Neuro-Oncology, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
Ramdas
L
L
Ke
S
S
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009036:Moths; D009994:Osmolar Concentration; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature
D011994:Recombinant Proteins; D008345:Manganese; D016392:Proto-Oncogene Proteins pp60(c-src); D008274:Magnesium