title: Expression of selenocysteine-containing glutathione S-transferase in eukaryote.
abstract: 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 chimaera 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/μmol 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.
authors.affiliation: College of Pharmaceutical Science, Jilin University, Changchun 130021, PR China.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Yin
authors.forename: Philip G
authors.initials: PG
authors.lastname: Board
authors.forename: Xiao
authors.initials: X
authors.lastname: Han
authors.forename: Zhenlin
authors.initials: Z
authors.lastname: Fan
authors.forename: Jingqi
authors.initials: J
authors.lastname: Fang
authors.forename: Zeyuan
authors.initials: Z
authors.lastname: Lu
authors.forename: Yini
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jingyan
authors.initials: J
authors.lastname: Wei
mesh_terms: D015153:Blotting, Western; D003001:Cloning, Molecular; D003062:Codon; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017279:Selenocysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D017279:Selenocysteine; D009249:NADP; C407705:GSTZ1 protein, human; D005982:Glutathione Transferase