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Recombinant human secretory phospholipase A 2 : Purification and characterization of the enzyme for active site studies
Author(s) -
Stadel J. M.,
Jones C.,
Livi G. P.,
Hoyle K.,
Kurdyla J.,
Roshak A.,
McLaughlin M. M.,
Pfarr D. A.,
Comer S.,
Strickler J.,
Bennett C. F.,
Marshall L.
Publication year - 1992
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.300050405
Subject(s) - chinese hamster ovary cell , recombinant dna , affinity chromatography , complementary dna , enzyme , biochemistry , microbiology and biotechnology , biology , chemistry , receptor , gene
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 anti‐inflammatory drugs, and to investigate further the role of PLA 2 in inflammatory disease.