PURIFICATION AND CHARACTERIZATION OF AN ACTIVATED FORM OF THE PROTEIN-TYROSINE KINASE LCK FROM AN ESCHERICHIA-COLI EXPRESSION SYSTEM

The lymphocyte-specific, nonreceptor protein tyrosine kinase Lck has b een purified from an Escherichia coli expression system using a monocl onal antibody column followed by dye-affinity chromatography. Polyacry lamide gel electrophoretic analysis of purified protein revealed a sin gle 56 kDa band, indicating that recombinant Lck was purified to near- homogeneity. The purified enzyme displayed tyrosine kinase activity as measured by both autophosphorylation and phosphorylation of exogenous substrates. Biochemical properties including protein phosphorylation and kinetic characteristics of the enzyme have been assessed. Peptide map analysis revealed that bacterially expressed Lck is phosphorylated predominantly on the autophosphorylation site (tyrosine-394), which i s characteristic for activated protein tyrosine kinases. Indeed, we fo und that the recombinant enzyme is approximately fivefold more active than Lck from resting T cells, which is extensively phosphorylated at the regulatory carboxy-terminal tyrosine residue (tyrosine-505). Thus, we have overproduced recombinant human Lck in E. coli and developed a simple two-step purification procedure which yields highly active enz yme. This will enable the identification and characterization of poten tial regulators and targets of Lck and thereby greatly facilitate stud ies which will clarify its role in T cell signal transduction. (C) 199 4 Wiley-Liss, Inc.