title: Purification and initial characterization of the lymphocyte-specific protein-tyrosyl kinase p56lck from a baculovirus expression system.
abstract: A baculovirus expression system has been used to express large quantities of the lymphocyte-specific protein-tyrosyl kinase p56lck. A series of chromatographic steps, including the novel application of metalchelate affinity chromatography for protein kinase purification, were employed to obtain p56lck in a highly active form. Recombinant p56lck was purified to apparent homogeneity as determined by polyacrylamide gel electrophoretic analyses and was found to migrate in SDS gels as two related species, both with apparent molecular masses close to 56 kDa. p56lck phosphorylated all assayed substrates exclusively on tyrosyl residues, and underwent autophosphorylation at one principal site, also on a tyrosyl residue. p56lck displayed a high affinity for a synthetic peptide corresponding to the cytoplasmic domain (residues 52-164) of the T-cell receptor zeta-chain (TCR-zeta) (Km approximately 6.5 microM) but a low affinity for a peptide corresponding to its own autophosphorylation site (Km approximately 900 microM). p56lck was also found to be highly active for a purified protein-tyrosyl kinase (Vmax greater than 400 pmol.min-1.micrograms-1 using the TCR-zeta (52-164) as a substrate). A variety of agents were tested for their ability to inhibit p56lck, with zinc ions (I50 approximately 1.7 mM) and staurosporine (I50 approximately 500 nM) proving the most potent.
authors.affiliation: Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
authors.forename: J D
authors.initials: JD
authors.lastname: Watts
authors.forename: G M
authors.initials: GM
authors.lastname: Wilson
authors.forename: E
authors.initials: E
authors.lastname: Ettenhadieh
authors.forename: I
authors.initials: I
authors.lastname: Clark-Lewis
authors.forename: C A
authors.initials: CA
authors.lastname: Kubanek
authors.forename: C R
authors.initials: CR
authors.lastname: Astell
authors.forename: J D
authors.initials: JD
authors.lastname: Marth
authors.forename: R
authors.initials: R
authors.lastname: Aebersold
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000201:enzymology*; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D010449:Peptide Mapping; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)