The cytoplasmic tyrosine kinase p72syk (SyK) plays an essential role in signaling via a variety of immune and nonimmune cell receptors, Syk is activated 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 baculovirus expression system. The enzyme was purified to 95% purity using a novel two-step affinity chromatography process using reactive yellow and phosphotyrosine columns. Yields of 3-10 mg purified Syk were obtained from 1 liter of infected insect cells, Western blotting, internal protein sequencing, and the specific tyrosine phosphorylation of a Syk peptide substrate indicated authenticity of the purified protein. The enzymatic properties of Syk were in good agreement with published data for the human enzyme, as the apparent 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 to the native protein isolated from B-cells such as autophosphorylation, Proteolytic cleavage of purified recombinant Syk was used to generate the kinase domain by mu-calpain, We therefore describe an efficient expression system and purification methodology to produce biologically active human Syk.