title: Heterologous expression and characterization of human cellular glutathione peroxidase mutants.
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-GPx1Secdisplayed 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-hGPx1Sershowed 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-hGPx1Sershowed 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.
authors.affiliation: College of Pharmaceutical Science, Jilin University, Changchun, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Guo
authors.affiliation: College of Pharmaceutical Science, Jilin University, Changchun, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.affiliation: College of Pharmaceutical Science, Jilin University, Changchun, China.
authors.forename: Xixi
authors.initials: X
authors.lastname: Liu
authors.affiliation: College of Pharmaceutical Science, Jilin University, Changchun, China.
authors.forename: Yinlong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: College of Pharmaceutical Science, Jilin University, Changchun, China.
authors.forename: Tuchen
authors.initials: T
authors.lastname: Guan
authors.affiliation: College of Pharmaceutical Science, Jilin University, Changchun, China.
authors.forename: Guiqiu
authors.initials: G
authors.lastname: Xie
authors.affiliation: College of Pharmaceutical Science, Jilin University, Changchun, China.
authors.forename: Jingyan
authors.initials: J
authors.lastname: Wei
keywords: cysteine auxotrophic strain; glutathione peroxidase; selenocystein; site-directed mutagenesis