RECOMBINANT PP60(C-SRC)FROM BACULOVIRUS-INFECTED INSECT CELLS - PURIFICATION AND CHARACTERIZATION

A simple and effective method has been developed to purify the recombi nant 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 activ ity of 3.9 mumol/min/mg protein using the substrate poly E4Y. This spe cific activity is many times higher than any published protocol. The e nzyme is stable for months when stored in buffered 10% glycerol at -70 -degrees-C. This purification technique is compared to the immuno-affi nity technique which is widely used for this enzyme. Enzyme kinetics w ere characterized with respect to substrate specificity, the effect of temperature, ionic strength, pH, and Mg+2 versus Mn+2 ions. Similar t o the enzyme expressed in human cells, the recombinant enzyme demonstr ated a higher Vmax and substrate specificity for poly E4Y over 5V-Agt- II. An activation energy of 14.2 kcal/mol was determined. Inhibition b y 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 phosp horylation of poly E4y, free Mg+2 was stimulatory while Mn+2 was inhib itory. In contrast, Mn+2 stimulated the phosphorylation of 5V-Agt-II.