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http://dx.doi.org/10.1007/s10059-009-0168-y

Expression of a Glutathione Reductase from Brassica rapa subsp. pekinensis Enhanced Cellular Redox Homeostasis by Modulating Antioxidant Proteins in Escherichia coli  

Kim, Il-Sup (Department of Biology, Kyungpook National University)
Shin, Sun-Young (Department of Biology, Kyungpook National University)
Kim, Young-Saeng (Department of Biology, Kyungpook National University)
Kim, Hyun-Young (Department of Biology, Kyungpook National University)
Yoon, Ho-Sung (Department of Biology, Kyungpook National University)
Publication Information
Molecules and Cells / v.28, no.5, 2009 , pp. 479-487 More about this Journal
Abstract
Glutathione reductase (GR) is an enzyme that recycles a key cellular antioxidant molecule glutathione (GSH) from its oxidized form (GSSG) thus maintaining cellular redox homeostasis. A recombinant plasmid to overexpress a GR of Brassica rapa subsp. pekinensis (BrGR) in E. coli BL21 (DE3) was constructed using an expression vector pKM260. Expression of the introduced gene was confirmed by semi-quantitative RT-PCR, immunoblotting and enzyme assays. Purification of the BrGR protein was performed by IMAC method and indicated that the BrGR was a dimmer. The BrGR required NADPH as a cofactor and specific activity was approximately 458 U. The BrGR-expressing E. coli cells showed increased GR activity and tolerance to $H_2O_2$, menadione, and heavy metal ($CdCl_2$, $ZnCl_2$ and $AlCl_2$)-mediated growth inhibition. The ectopic expression of BrGR provoked the co-regulation of a variety of antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. Consequently, the transformed cells showed decreased hydroperoxide levels when exposed to stressful conditions. A proteomic analysis demonstrated the higher level of induction of proteins involved in glycolysis, detoxification/oxidative stress response, protein folding, transport/binding proteins, cell envelope/porins, and protein translation and modification when exposed to $H_2O_2$ stress. Taken together, these results indicate that the plant GR protein is functional in a cooperative way in the E. coli system to protect cells against oxidative stress.
Keywords
antioxidant enzymes; Brassica rapa subsp. pekinensis; Escherichia coli; glutathione reductase; stress tolerance;
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