Recombinant human secretory phospholipase A2: purification and characterization of the enzyme for active site studies.
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.
Stadel
J M
JM
Department of Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
Jones
C
C
Livi
G P
GP
Hoyle
K
K
Kurdyla
J
J
Roshak
A
A
McLaughlin
M M
MM
Pfarr
D A
DA
Comer
S
S
Strickler
J
J
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D010741:Phospholipases A / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D010920:Placenta / Q000201:enzymology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2