Baculovirus expression and purification of the second messenger enzyme phospholipase C-gamma 1, a tyrosine kinase substrate.

A rat cDNA encoding phospholipase C-gamma 1 (PLC-gamma 1) was expressed as a histidine-tagged fusion protein in insect cells utilizing the expression vector pBlueBacHis. The fusion protein was purified by Ni(2+)-agarose affinity chromatography to apparent homogeneity as defined by SDS-PAGE and Coomassie staining. Using a Triton X-100/PIP2 mixed micelle assay, (His6)-PLC-gamma 1 exhibited a calcium-dependent specific enzyme activity of 3 mumol/min/mg, a value similar to that reported for purified bovine brain PLC-gamma 1. Also similar to bovine brain PLC-gamma 1, (His6)-PLC-gamma 1 activity was stimulated by phosphatidic acid and inhibited by adenosine 5'monophosphate. (His6)-PLC-gamma 1 interacted directly with two known PLC-gamma 1 binding proteins, dynamin and the activated EGF receptor. Also, purified (His6)-PLC-gamma 1 was a tyrosine phosphorylation substrate for purified EGF receptor. These results suggest that (His6)-PLC-gamma 1 can be overexpressed as a functional enzyme in baculovirus-infected insect cells and purified by a one-step metal affinity chromatography procedure.