ACTIVE RECOMBINANT HUMAN CYTOSOLIC PHOSPHOLIPASE A(2) IS EXPRESSED INESCHERICHIA-COLI

The cDNA encoding human cytosolic phospholipase A(2) (cPLA(2)) has bee n subcloned into a prokaryotic pET16b expression vector which also enc odes an amino-terminal deca-histidine affinity tag to facilitate purif ication of the recombinant enzyme. Soluble, active fusion protein, des ignated His-cPLA(2), has been obtained reproducibly from this expressi on system using the E. coli strain BL21 (DE3). The protein has been pu rified to homogeneity in four steps and the mass confirmed by electros pray mass spectrometry. His-cPLA(2) was characterized by kinetic analy sis which demonstrated that the enzyme is similar to native cPLA(2) in all respects investigated. Specifically, the enzyme binds to anionic vesicles containing substrate, and acts processively on these vesicles . Enzymatic activity is supported by the presence of Ca2+ and several other divalent metal ions, and is inhibited by several transition meta l ions. Finally, the enzyme demonstrates lysophospholipase activity an d exhibits a high selectivity for sn-2 arachidonyl esters. This prokar yotic expression system yields moderate amounts of unmodified recombin ant His-cPLA(2), and is advantageous for rapid production of protein a nd mutational analyses. (C) 1995 Academic Press, Inc.