JOURNAL OF MOLECULAR RECOGNITION, VOL. 5 , 145-153 (1992)
Recombinant Human Secretory Phospholipase A,: Purification and Characterization of the Enzyme for Active Site Studies J. M. Stadel,* C. Jones,? G. P. Livi,$ K. Hoyle, J. Kurdyla,? A. Roshak, M. M. McLaughlin,$ D. A. Pfarr,$ S. Comer,$ J. Strickler,?? C. F. Bennett$$ and L. Marshall Departments of Pharmacology, tProtein Biochemistry, $Gene Expression Sciences, PBiopharmaceutical Manufacturing and ttMacromolecular Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA
A secreted form of phospholipase Az (PLAJ is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLAz was cloned from a human placental cDNA library. The cDNA encoding the human PLAz 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 PLAZinto 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(viny1idenedifluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLAz activity was 58%. A direct comparison between the purified recombinant human PLAz and PLAz purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLAz inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLAz can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLAz inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLAz in inflammatory disease.
INTRODUCTION
Phospholipases A, (PLA,, EC 3.1.1.4) represent a family of enzymes that specifically catalyze the liberation of free fatty acids from the sn-2 position of phospholipids. The products of this hydrolysis, free fatty acid in the form of arachidonic acid and lysophospholipids, are pro-inflammatory lipid precursors. Arachidonic acid is further metabolized by cyclooxygenase and lipoxygenase enzymes to form eicosanoids. Lysophospholipids may themselves promote inflammation or they can be converted by acetylation to PAF. Thus, stimulation of PLA, activity results in the production of a variety of lipid derived pro-inflammatory mediators (Dennis, 1990; Kaiser et al., 1990; Marshall and Chang, 1990; Filep, 1991). Biochemically, the soluble/extracellular PLA,s share a number of properties. They are of relatively low molecular weight (
Recombinant Human Secretory Phospholipase A,: Purification and Characterization of the Enzyme for Active Site Studies J. M. Stadel,* C. Jones,? G. P. Livi,$ K. Hoyle, J. Kurdyla,? A. Roshak, M. M. McLaughlin,$ D. A. Pfarr,$ S. Comer,$ J. Strickler,?? C. F. Bennett$$ and L. Marshall Departments of Pharmacology, tProtein Biochemistry, $Gene Expression Sciences, PBiopharmaceutical Manufacturing and ttMacromolecular Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA
A secreted form of phospholipase Az (PLAJ is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLAz was cloned from a human placental cDNA library. The cDNA encoding the human PLAz 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 PLAZinto 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(viny1idenedifluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLAz activity was 58%. A direct comparison between the purified recombinant human PLAz and PLAz purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLAz inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLAz can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLAz inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLAz in inflammatory disease.
INTRODUCTION
Phospholipases A, (PLA,, EC 3.1.1.4) represent a family of enzymes that specifically catalyze the liberation of free fatty acids from the sn-2 position of phospholipids. The products of this hydrolysis, free fatty acid in the form of arachidonic acid and lysophospholipids, are pro-inflammatory lipid precursors. Arachidonic acid is further metabolized by cyclooxygenase and lipoxygenase enzymes to form eicosanoids. Lysophospholipids may themselves promote inflammation or they can be converted by acetylation to PAF. Thus, stimulation of PLA, activity results in the production of a variety of lipid derived pro-inflammatory mediators (Dennis, 1990; Kaiser et al., 1990; Marshall and Chang, 1990; Filep, 1991). Biochemically, the soluble/extracellular PLA,s share a number of properties. They are of relatively low molecular weight (
