Purification and characterization of human Syk produced using a Baculovirus expression system

The cytoplasmic tyrosine kinase p72syk (SyK) plays an essential role in sig naling via a variety of immune and nonimmune cell receptors, Syk is activat ed in response to the engagement of the appropriate cell surface receptors and can phosphorylate downstream targets and recruit additional SH2-domain- containing proteins. In order to study the characteristics of Syk in vitro, we have overexpressed untagged, full-length human Syk in a recombinant bac ulovirus expression system. The enzyme was purified to 95% purity using a n ovel two-step affinity chromatography process using reactive yellow and pho sphotyrosine columns. Yields of 3-10 mg purified Syk were obtained from 1 l iter of infected insect cells, Western blotting, internal protein sequencin g, and the specific tyrosine phosphorylation of a Syk peptide substrate ind icated authenticity of the purified protein. The enzymatic properties of Sy k were in good agreement with published data for the human enzyme, as the a pparent K-m of Syk for ATP was 10 mu M and the peptide substrate was 3 mu M . The recombinant protein also showed similar biochemical characteristics t o the native protein isolated from B-cells such as autophosphorylation, Pro teolytic cleavage of purified recombinant Syk was used to generate the kina se domain by mu-calpain, We therefore describe an efficient expression syst em and purification methodology to produce biologically active human Syk. ( C) 2000 Academic Press.