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Overexpression and Purification of the Antioxidant Selenoprotein Plasma Glutathione Peroxidase
Author(s) -
Ottaviano Filomena G.,
Handy Diane E.,
Loscalzo Joseph
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a649-b
Subject(s) - selenocysteine , selenoprotein , biochemistry , gpx3 , microbiology and biotechnology , glutathione peroxidase , chemistry , stop codon , gpx4 , mutant , gpx1 , glutathione , biology , amino acid , cysteine , enzyme , gene
Plasma glutathione peroxidase (GPx‐3) is a selenocysteine‐containing protein that catalyzes the reduction of hydrogen peroxide and lipid hydroperoxides using glutathione as a reducing agent (GSH). Our goals are to overexpress GPx‐3, determine its activity, and study its regulation. The complex expression of selenoproteins requires specific translational cofactors (SBP2, SelD, and tRNA Sec ), adequate organified selenium, and the selenocysteine insertion sequence (SECIS), which facilitate read‐through of the UGA codon for selenocysteine (Sec) and incorporation in the growing polypeptide, rather than premature termination of translation. GPx‐3 cDNA and the SECIS were cloned into a pcDNA 4/V5 His C vector. This vector contains a unique C terminal His tag important for purification of GPx‐3. Stable Cos 7 cell lines of the wild type, SECIS containing mutant 3′UTR fragments, and a Sec → Cys mutant were also generated and grown in adequate sodium selenite to ensure optimal organification and selenocysteine synthesis. mRNA expression of endogenous and recombinant forms of GPx‐3 were detected by RT‐PCR in Cos 7 cells. Immunoprecipitation with anti His C term antibody followed by Western blot with anti V5 antibody showed detection of both the wild type and the selenocysteine UGA mutant protein. Mutating the UGA codon of GPx‐3 allows for translational read‐through without the requirement for translational cofactors. Support: NIH HL61795, HL58976, PO1 HL81587