Purification of baculovirus-overexpressed cytosolic phospholipase A2 using a single-step affinity column chromatography.
Cytosolic phospholipase A2 (cPLA2) plays a key role in the production of proinflammatory lipid mediators such as prostaglandins, thromboxane, and leukotrienes. cPLA2, an arachidonic acid-selective, 85-kDa protein has been purified, cloned, and partially characterized from a number of tissues. However, the purification schemes previously published by several groups are lengthy, involving several chromatographic steps and resulting in low yields of enzyme. Here we report the preparation of a novel affinity column (Affi-656) by immobilizing a competitive inhibitor of cPLA2, and a single-step purification of this enzyme. This column selectively retains cPLA2 activity from the cytosolic fractions of Sf9 cells infected with recombinant baculovirus which is eluted by a gradient of CHAPS in the elution buffer. Purification of cPLA2 to homogeneity can thus be accomplished in a single step. Moreover, mutant cPLA2 (Ser505/Ala505) which is no longer phosphorylated at Ser505 is also retained on Affi-656; mutation at this residue does not disrupt its binding to the affinity column. To our knowledge, this is the first report of cPLA2 affinity purification. Affi-656 is a convenient, reproducible, and high-capacity affinity column, and is a valuable tool for rapid purification of cPLA2 in large quantities.
Abdullah
K M
KM
Department of Biochemistry, Merck Frosst Center for Therapeutic Research, Point Claire-Dorval, Quebec, Canada.
Leger
S
S
Perrier
H
H
Cromlish
W A
WA
Kennedy
B
B
Gresser
M
M
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D003600:Cytosol / Q000201:enzymology; D015151:Immunoblotting; D010741:Phospholipases A / Q000235:genetics / Q000302:isolation & purification*; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology / Q000821:virology
D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2