Purification and characterization of recombinant microsomal prostaglandin E synthase-1
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.
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