Abstract
A secreted form of phospholipase A~2~ (PLA~2~) is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLA~2~ was cloned from a human placental cDNA library. The cDNA encoding the human PLA~2~ was subcloned into an expression vector and subsequently transfected into Chinese hamster ovary (CHO) cells. A stable CHO cell clone, secreting ca I mg/L of recombinant PLA~2~ into the medium, was scaled up in culture to 180L. 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 PLA~2~ activity was 58%. A direct comparison between the purified recombinant human PLA~2~ and PLA~2~ purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to know PLA~2~ inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLA~2~ can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLA~2~ inhibitors as potential antiβinflammatory drugs, and to investigate further the role of PLA~2~ in inflammatory disease.