PURIFICATION AND CHARACTERIZATION OF RECOMBINANT HUMAN P50(CSK) PROTEIN-TYROSINE KINASE FROM AN ESCHERICHIA-COLI EXPRESSION SYSTEM OVERPRODUCING THE BACTERIAL CHAPERONES GROES AND GROEL

An Escherichia coil expression system overproducing the bacterial chap erones GroES and GroEL was engineered and has been successfully used t o produce large quantities of the recombinant human protein-tyrosine k inase p50(csk). The co-overproduction of the two chaperones with p50(c sk) results in increased solubility of the kinase and allows purificat ion of milligram amounts of active enzyme. Analysis of the purified pr otein by SDS/polyacrylamide gel electrophoresis reveals a single band with an apparent molecular mass of 50 kDa, indicating that recombinant human p50(csk) has been purified to near homogeneity. The purified en zyme displays tyrosine kinase activity as measured by both autophospho rylation and phosphorylation of exogenous substrates. Biochemical prop erties, including in vitro substrate specificity and enzymatic charact eristics of the enzyme, have been assessed and compared with those of members of the Src family of protein-tyrosine kinases. Results indicat e that p50(csk) and p56(lck) have different substrate specificities an d that p50(csk) and p60(c-src) have similar kinetic parameters. The su ccessful production and purification of an enzymatically active form o f p50(csk) will enable further characterization of this important kina se and allow clarification of its physiological role. In addition, the results suggest that the approach described may be generally applicab le to improve the solubility of recombinant proteins which otherwise a re produced in an insoluble form in E. coil.