Hj. George et al., EXPRESSION PURIFICATION AND CHARACTERIZATION OF RECOMBINANT HUMAN INDUCIBLE PROSTAGLANDIN G H SYNTHASE FROM BACULOVIRUS-INFECTED INSECT CELLS/, Protein expression and purification, 7(1), 1996, pp. 19-26
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