Recombinant human secretory phospholipase A₂: Purification and characterization of the enzyme for active site studies

Abstract

A secreted form of phospholipase A2 (PLA2) is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLA2 was cloned from a human placental cDNA library. The cDNA encoding the human PLA2 was subcloned into an expression vector and subsequently transfected into Chinese hamster ovary (CHO) cells. A stable CHO cell clone, secreting ca 1 mg/L of recombinant PLA2 into the medium, was scaled up in culture to 180 L. The recombinant enzyme was purified from the cell supernatant to apparent homogeneity by a novel procedure combining adsorption to poly(vinylidene difluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLA2 activity was 58%. A direct comparison between the purified recombinant human PLA2 and PLA2 purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLA2 inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLA2 can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLA2 inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLA2 in inflammatory disease.

Cytosolic 85-kDa Phospholipase A2-mediated Release of Arachidonic Acid Is Critical for Proliferation of Vascular Smooth Muscle Cells

“…Recombinant human Type II 14-kDa PLA 2 (rh Type II 14-kDa PLA 2 ) was cloned from a human placenta library, expressed, and purified as described previously (52,53). Recombinant human cPLA 2 (rh 85-kDa PLA 2 ) was prepared using a U937 85-kDa PLA 2 cDNA subcloned into the baculovirus vector pAcCL29 and expression in Spotoptera frugiperde (SF21) cells as described previously (54 (14).…”

β-Lactams SB 212047 and SB 216754 Are Irreversible, Time-Dependent Inhibitors of Coenzyme A-Independent Transacylase

“…PLA 2 activities were determined using [ 3 H]arachidonate-labeled Escherichia coli membranes by measuring the liberation of free [ 3 H]AA (Marshall and McCarte-Roshak, 1992). Human type II, 14-kDa PLA 2 was purified from a clone expressed in Chinese hamster ovary cells (Stadel et al, 1992). Cytosolic 85-kDa PLA 2 (Kramer et al, 1991) was obtained from a clone expressed in baculovirus-infected insect cells (Amegazie et al, 1993).…”

Human Keratinocytes Possess an sn-2 Acylhydrolase that is Biochemically Similar to the U937-Derived 85-kDa Phospholipase A2

“…Recombinant human (rh) type II 14-kDa PLAj cloned from placenta mRNÂ as expressed as an authentic processed enzyme in CHO cells and purified gssentially by literature methods with some modifications as previously described [22,23]. The purified enzyme had a specific activity of 200-300 umol/mg/min in an PH]-AA Escherichia coti assay and was hiochemically indistinguishable from the enzyme purified from human synovial fluid [23].…”

“…The purified enzyme had a specific activity of 200-300 umol/mg/min in an PH]-AA Escherichia coti assay and was hiochemically indistinguishable from the enzyme purified from human synovial fluid [23]. \J937 85-kDa PLA2 was purified from the cytosol of the human leukemic nionoblast U937 cell line as previously described [24].…”