title: Characterization of recombinant human gastrointestinal glutathione peroxidase mutant produced in Escherichia coli.
abstract: Gastrointestinal glutathione peroxidase (GI-GPx, GPx2) is a selenium-dependent enzyme and regarded as the first line of defense against oxidative stress caused by ingested pro-oxidants or gut microbes. As the essential part of the catalytic site of GPx2, selenocysteine (Sec) is encoded by an in-frame UGA stop codon, which makes the expression of human GPx2 (hGPx2) using traditional recombinant DNA technology difficult. In order to produce bioactive recombinant hGPx2, the gene of hGPx2 was designed with the conversion of the codons for four cysteine (Cys) residues to the codons for serine (Ser) residues and the codon for Sec-40 was changed to the codon for Cys. This recombinant seleno-hGPx2 mutant was obtained using a single protein production system in a cysteine (Cys) auxotrophic strain, in which Sec was introduced into the protein via tRNA(Cys) misleading. The activity of this mutant was in the same order of magnitude as that of hGPx4, but about one order of magnitude lower than that of hGPx1 and hGPx3. Further study showed that the mutant exhibited pH and temperature optima of 7.4 and 25°C, respectively. The results obtained from the kinetic analysis demonstrated that it followed a typical ping-pong mechanism similar to native GPx. As there was no report on the activity of purified GPx2, this research was valuable in recognizing native GPx2. In addition, a three-dimensional structure of seleno-hGPx2 mutant was constructed, which could facilitate further analysis of the role and the catalytic mechanism of native GPx2.
authors.affiliation: College of Pharmaceutical Science, Jilin University , Changchun , P. R. China.
authors.forename: X
authors.initials: X
authors.lastname: Guo
authors.forename: J
authors.initials: J
authors.lastname: Song
authors.forename: T
authors.initials: T
authors.lastname: Guan
authors.forename: S
authors.initials: S
authors.lastname: Wang
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: Y
authors.initials: Y
authors.lastname: Meng
authors.forename: J
authors.initials: J
authors.lastname: Guo
authors.forename: T
authors.initials: T
authors.lastname: Li
authors.forename: Chuang
authors.initials: C
authors.lastname: Ma
authors.forename: J
authors.initials: J
authors.lastname: Wei
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D005979:Glutathione Peroxidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051140:Selenoproteins / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D051140:Selenoproteins; C117436:GPX2 protein, human; D005979:Glutathione Peroxidase
keywords: antioxidant; glutathione peroxidase; selenocysteine; selenoprotein; single protein production system