The search for isoform-specific enzyme inhibitors has been the focus of much recent research effort. Towards this goal, human recombinant cyclooxygenase-2 (EC 1.14.99.1, prostaglandin H synthase) was expressed in insect cells and purified to > 98% purity. Recombinant enzyme was characterized both by physical methods and activity measurements and shown to be fully active with kinetic properties similar to native COX-2 and COX-1. After detergent extraction, the enzyme had hydrodynamic properties indistinguishable from native bovine COX-1 and corresponded to the enzyme dimer as measured with size-exclusion chromatography. Peptide mapping via Lys-C protease identified a site of N-linked glycosylation and the aspirin covalent modification site. In the presence of heme, aspirin-specifically acetylated Ser-516. The enzyme will be suitable for biophysical studies and may lead to isoform-specific enzyme inhibitors.
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