PURIFICATION, CHARACTERIZATION AND SELECTIVE-INHIBITION OF HUMAN PROSTAGLANDIN-G H SYNTHASE-1 AND SYNTHASE-2 EXPRESSED IN THE BACULOVIRUS SYSTEM/

Human prostaglandin G/H synthase 1 and 2 were expressed in the baculov irus expression system and purified to high levels. Both enzymes were glycosylated. PGHS-1 appeared to be homogeneous by SDS-PAGE analysis b ut two closely migrating bands were detected in PGHS-2 preparation whi ch were evidently due to heterogeneity in glycosylation. The amino-aci d sequence of the N-termini of both isoforms indicated that the signal sequences were efficiently cleaved by the insect cells. The recombina nt human PGHS-1 and PGHS-2 possessed both cyclooxygenase and peroxidas e activities. Both had high affinities for arachidonate as substrate a nd underwent self-inactivation during catalysis. The recombinant isofo rms were not pharmacologically identical, since some NSAIDs were selec tive inhibitors of either PGHS-1 or PGHS-2. This is the first report o f high levels of expression and purification of human PGHS isoforms. T he recombinant enzymes are invaluable in developing potent PGHS-2 sele ctive inhibitors that may be efficacious anti-inflammatory drugs with no or low levels of toxicity.