EXPRESSION PURIFICATION AND CHARACTERIZATION OF RECOMBINANT HUMAN INDUCIBLE PROSTAGLANDIN G H SYNTHASE FROM BACULOVIRUS-INFECTED INSECT CELLS/

The inducible isoform of human prostaglandin G/H synthase (human cyclo oxygenase; hCOX2) has been produced in Sf21 insect cells using the bac ulovirus expression system. The full-length gene for hCOX2 was placed under the control of the hybrid pCap/PolH promoter and recombinant vir us generated by homologous recombination. Insect cells infected with r ecombinant virus synthesized active hCOX2 at levels exceeding 5% of to tal cellular protein 72 h postinfection. Optimal production on a prepa rative scale and high activity yields were attained in 8-liter spinner flasks using a supplemented Grace's medium containing 10% FCS. The ap e-enzyme was purified to homogeneity by detergent extraction and ion e xchange chromatography and functionally reconstituted with heme to for m the hole-enzyme. The purified enzyme from insect cells was identifie d as hCOX2 by enzymatic activity, Western immunoassay, and N-terminal sequence analysis; the latter also indicated correct processing of the hCOX2 signal sequence. Insect recombinant hCOX2 displays high specifi c activity for both cyclooxygenase and peroxidase activities at levels indistinguishable from mammalian derived enzyme. Spectroscopic analys is suggests that the recombinant enzyme adopts native-like secondary a nd tertiary structure. The data presented here demonstrate that this s ystem is capable of providing high yields of active enzyme for biochem ical, biophysical, and pharmacological investigations. (C) 1996 Academ ic Press, Inc