Purification and characterization of recombinant microsomal prostaglandin E synthase-1

https://doi.org/10.1016/S1046-5928(02)00566-1Get rights and content

Abstract

Recombinant human microsomal prostaglandin E2 synthase-1 (mPGES-1) was expressed in a baculovirus-Sf9 cell system. The mPGES-1 was solubilized from Sf9 cell membranes with diheptanoylphosphatidylcholine and purified in the presence of octylglucoside using hydroxyapatite column chromatography. The Km values of the substrates PGH2 and GSH were 14μM and 0.75 mM, respectively, with the purified enzyme. The specific activity (4μmol/min/mg) was increased 3–5-fold by non-ionic and zwitterionic detergents. Kinetic analysis showed that dodecylmaltoside increases Vmax but does not affect the Km values of either substrate. Several other thiol-containing compounds were tested as glutathione replacements, none of which yielded detectable enzyme activity. During enzyme catalysis, glutathione was not oxidized and therefore can be considered an enzyme cofactor. No glutathione transferase or peroxidase activity could be determined with a range of potential substrates. The results show that purified mPGES-1 has a specific activity similar to Cox-2, consistent with its postulated role in Cox-2 mediated PGE2 formation.

Section snippets

Baculovirus expression of mPGES-1

The sequence of mPGES-1 is presented in GenBank as Accession No. AF027740. A vector (pT7T3D) containing the full length sequence was obtained from the Washington University-Merck EST project and purchased from Research Genetics (Huntsville, AL). The sequence containing the open reading frame of mPGES-1 was digested with EcoRI/NotI from pT7T3D and subcloned into the EcoRI/NotI multiple cloning site of pFastBac vector (Invitrogen). The virus was obtained by use of the Bac-to-Bac expression system

Results and discussion

The cDNA insert of mPGES-1 (GenBank: AF027740) was subcloned into the pFastBac plasmid and the bacmid preparation and isolation were performed in bacteria. Sf9 cells were transfected with bacmid DNA containing the mPGES-1 cDNA insert and viral stocks were prepared and amplified. Mock bacmid DNA, which did not contain the mPGES-1 cDNA, was also prepared. The viral stock was used to perform a time course analysis of mPGES-1 expression. Sf9 cells were infected with recombinant baculovirus and the

Acknowledgements

The authors thank Gillian Greig for supplying PGD synthase.

Cited by (50)

  • Eicosanoids and Renal Function

    2013, Seldin and Geibisch's The Kidney
  • Eicosanoids and Renal Function

    2012, Seldin and Giebisch's The Kidney: Physiology and Pathophysiology
  • Pyrrole alkanoic acid derivatives as nuisance inhibitors of microsomal prostaglandin E <inf>2</inf> synthase-1

    2012, European Journal of Medicinal Chemistry
    Citation Excerpt :

    One critical factor in these assays is the chemical instability of PGH2, which degrades in aqueous solutions non-enzymatically into the enzyme product PGE2 and into PGD2 with a half life of about 10 min at room temperature [42]. To minimize non-enzymatic PGE2 formation, short incubation times (30 s–5 min) and low temperatures (0–20 °C) are generally applied [10,20,22,24–34,37,43–49]. At the end of the enzyme reaction unreacted PGH2 is converted to PGF2α by addition of SnCl2 or FeCl2.

View all citing articles on Scopus
View full text