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Expression and characterization of recombinant human phospholipid hydroperoxide glutathione peroxidase
Author(s) -
Han Xiao,
Fan Zhenlin,
Yu Yang,
Liu Shaoli,
Hao Yazhou,
Huo Rui,
Wei Jingyan
Publication year - 2013
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1002/iub.1220
Subject(s) - phospholipid hydroperoxide glutathione peroxidase , gpx4 , gpx1 , gpx6 , gpx3 , biochemistry , recombinant dna , chemistry , microbiology and biotechnology , blot , transfection , glutathione , affinity chromatography , chinese hamster ovary cell , peroxidase , hek 293 cells , cytosol , glutathione peroxidase , biology , enzyme , gene , receptor
Phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx4; EC1.11.1.12) is a selenoperoxidase that can directly reduce phospholipid and cholesterol hydroperoxides. The mature cytoplasmic GPx4 is a monomeric protein with molecular weight of 19.5 kDa. In this study, human GPx4 ( hGPx4 ) gene was amplified from the complementary DNA library of human hepatoma cell line. Eukaryotic expression plasmid pSelExpress1‐leader‐GPx4 was constructed and transfected into the eukaryotic cells HEK293T. Expression of hGPx4 was detected by Western blotting, and the target protein was purified by immobilized metal affinity chromatography. The results of the activity and kinetics of the purified protein show that the obtained protein follows a “ping–pong” mechanism, which is similar to that of native cytosolic glutathione peroxidase (GPx1; EC1.11.1.9). This is the first time that hGPx4 could be expressed and purified from HEK293T cells, and this work will provide an important resource of hGPx4 for its functional study in vitro and in vivo . © 2013 IUBMB Life, 65(11):951–956, 2013