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Keywords:

  • glutathione peroxidase;
  • selenocystein;
  • cysteine auxotrophic strain;
  • site-directed mutagenesis

Abstract

Cellular glutathione peroxidase (GPx1; EC1.11.1.9) is a major intracellular antioxidant selenoenzyme in mammals. However, the complicated expression mechanism of selenocysteine (Sec)-containing protein increases the difficulty of expressing human GPx1 (hGPx1) in Escherichia coli (E. coli). In this study, hGPx1 gene was cloned from a cDNA library of the human hepatoma cell line HepG2. The codon UGA encoding Sec49 of hGPx1 was first mutated to UGC encoding cysteine (Cys) and then biosynthetically converted to Sec during expression in an E. coli BL21(DE3)cys auxotrophic system. Seleno-GPx1Sec displayed a low GPx activity of 522 U/μmol. To improve the activity, the other five Cys residues (C2, C78, C115, C156, C202) were mutated to serine (Ser) in one hGPx1 molecule. The mutant seleno-hGPx1Ser showed a high activity of 5278 U/μmol, which was more than 10-fold enhanced as compared with seleno-GPx1Sec. The activity was the highest among all of those seleno-proteins obtained by this method so far. Kinetic analysis of seleno-hGPx1Ser showed a typical ping-pong mechanism, which was similar to those of natural GPxs. This research will be of value in overcoming the problem of limited sources of natural GPx and substantially promotes the research of the characterization of GPx. © 2014 IUBMB Life, 66(3):212–219, 2014