Glutathione peroxidase (GPX) is a crucial antioxidant selenocysteine (Sec) containing enzyme which plays a significant role in protecting cells against oxidative damage by catalyzing the reduction of hydroperoxides with glutathione (GSH). Several methods have been used to generate GPX mimics, however, only a few of these methods involved genetic engineering and none of them have achieved specific site-directed incorporation of Sec without other modifications, which has hampered further structure-function studies. Here, we report for the first time the conversion of human glutathione transferase Zeta (hGSTZ1-1) into seleno-hGSTZ1-1 by means of genetic engineering in eukaryotes. Fluorescence microscopy images of the expression of Seleno-GST-green fluorescent protein chirnaera indicated that we successfully achieved the read-through of the UGA codon to specifically incorporate Sec. Therefore, we achieved the conversion of human glutathione transferase Zeta (hGSTZ1-1) into a seleno-GST (seleno-hGSTZ1-1) by means of genetic engineering in eukaryotes. These results show that recombinant selenoproteins with incorporation of specific selenocysteine residues may be heterologously produced in eukaryotes by using a Sec insertion sequence in the 3' untranslated region (3'-UTR) of the mRNA, and the recombinant selenoproteins is single catalytically active residue and well-characterized structure. In this case a novel GPX activity of 2050 +/- 225 U/mu mol was introduced into hGSTZ1-1 by substitution of serine 15 by Sec 15. This result will lay a foundation for preparing much smaller GPX mimics with higher activity. (c) 2012 Elsevier Inc. All rights reserved.