Expression purification and characterization of recombinant human inducible prostaglandin G/H synthase from baculovirus-infected insect cells

Protein Expression and Purification
H J GeorgeR A Copeland

Abstract

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-lik...Continue Reading

Citations

Jan 19, 2006·Molecular and Cellular Endocrinology·Trevor M PenningHseuh-Kung Lin
Sep 13, 2002·Biotechnology and Bioengineering·Colleen SicoAmit Varma
Nov 20, 2003·The Journal of Biological Chemistry·Bijan BambaiRichard J Kulmacz
May 7, 1999·Bioorganic & Medicinal Chemistry Letters·D J PintoR L Magolda
Dec 10, 1997·Journal of Chromatography. B, Biomedical Sciences and Applications·J Evangelista Dyr, J Suttnar
Dec 1, 2004·Journal of Natural Products·Lawrence M Szewczuk, Trevor M Penning
Sep 5, 2009·Archives of Biochemistry and Biophysics·Ah-Lim Tsai, Richard J Kulmacz

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