PURIFICATION OF BACULOVIRUS-OVEREXPRESSED CYTOSOLIC PHOSPHOLIPASE A(2) USING A SINGLE-STEP AFFINITY COLUMN CHROMATOGRAPHY

Cytosolic phospholipase A(2) (cPLA(2)) plays a key role in the production of proinflammatory lipid mediators such as prostaglandins, thromboxane, and leukotrienes. cPLA(2), an arachidonic acid-selective, 85-kDa protein has been purified, cloned, and partially characterized from a number of tissues. However, the purification schemes previously published by several groups are lengthy, involving several chromato-graphic steps and resulting in low yields of enzyme. Here we report the preparation of a novel affinity column (Affi-656) by immobilizing a competitive inhibitor of cPLA(2), and a single-step purification of this enzyme. This column selectively retains cPLA(2) activity from the cytosolic fractions of Sf9 cells infected with recombinant baculovirus which is eluted by a gradient of CHAPS in the elution buffer. Purification of cPLA(2) to homogeneity can thus be accomplished in a single step. Moreover, mutant cPLA(2) (Ser(505)/Ala(505)) which is no longer phosphorylated at Ser(505) is also retained on Affi-656; mutation at this residue does not disrupt its binding to the affinity column. To our knowledge, this is the first report of cPLA(2) affinity purification. Affi-656 is a convenient, reproducible, and high-capacity affinity column, and is a valuable tool for rapid purification of cPLA(2) in large quantities. (C) 1995 Academic Press, Inc.