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 cyclooxygenase; hCOX2) has been produced in Sf21 insect cells using the baculovirus expression system. The full-length gene for hCOX2 was placed under the control of the hybrid pCap/PolH promoter and recombinant virus generated by homologous recombination. Insect cells infected with recombinant virus synthesized active hCOX2 at levels exceeding 5% of total cellular protein 72 h postinfection. Optimal production on a preparative scale and high activity yields were attained in 8-liter spinner flasks using a supplemented Grace's medium containing 10% FCS. The apo-enzyme was purified to homogeneity by detergent extraction and ion exchange chromatography and functionally reconstituted with heme to form the holo-enzyme. The purified enzyme from insect cells was identified 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 specific activity for both cyclooxygenase and peroxidase activities at levels indistinguishable from mammalian derived enzyme. Spectroscopic analysis suggests that the recombinant enzyme adopts native-like secondary and tertiary structure. The data presented here demonstrate that this system is capable of providing high yields of active enzyme for biochemical, biophysical, and pharmacological investigations.
George
H J
HJ
DuPont Merck Research Laboratories, Wilmington, Delaware 19880-0400, USA.
Van Dyk
D E
DE
Straney
R A
RA
Trzaskos
J M
JM
Copeland
R A
RA
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D010544:Peroxidases / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D018411:Spodoptera; D013772:Tetramethylphenylenediamine / Q000378:metabolism
D011994:Recombinant Proteins; D016718:Arachidonic Acid; D010544:Peroxidases; D011451:Prostaglandin-Endoperoxide Synthases; D013772:Tetramethylphenylenediamine