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Expression of a glutathione reductase from Brassica rapa subsp. pekinensis enhanced cellular redox homeostasis by modulating antioxidant proteins in Escherichia coli.
Mol Cells. 2009 Nov 30; 28(5):479-87.MC

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 semiquantitative 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(2)O(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(2)O(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.

Authors+Show Affiliations

Department of Biology, Kyungpook National University, Daegu 702-701, Korea.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19936628

Citation

Kim, Il-Sup, et al. "Expression of a Glutathione Reductase From Brassica Rapa Subsp. Pekinensis Enhanced Cellular Redox Homeostasis By Modulating Antioxidant Proteins in Escherichia Coli." Molecules and Cells, vol. 28, no. 5, 2009, pp. 479-87.
Kim IS, Shin SY, Kim YS, et al. Expression of a glutathione reductase from Brassica rapa subsp. pekinensis enhanced cellular redox homeostasis by modulating antioxidant proteins in Escherichia coli. Mol Cells. 2009;28(5):479-87.
Kim, I. S., Shin, S. Y., Kim, Y. S., Kim, H. Y., & Yoon, H. S. (2009). Expression of a glutathione reductase from Brassica rapa subsp. pekinensis enhanced cellular redox homeostasis by modulating antioxidant proteins in Escherichia coli. Molecules and Cells, 28(5), 479-87. https://doi.org/10.1007/s10059-009-0168-y
Kim IS, et al. Expression of a Glutathione Reductase From Brassica Rapa Subsp. Pekinensis Enhanced Cellular Redox Homeostasis By Modulating Antioxidant Proteins in Escherichia Coli. Mol Cells. 2009 Nov 30;28(5):479-87. PubMed PMID: 19936628.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Expression of a glutathione reductase from Brassica rapa subsp. pekinensis enhanced cellular redox homeostasis by modulating antioxidant proteins in Escherichia coli. AU - Kim,Il-Sup, AU - Shin,Sun-Young, AU - Kim,Young-Saeng, AU - Kim,Hyun-Young, AU - Yoon,Ho-Sung, Y1 - 2009/11/18/ PY - 2009/09/26/received PY - 2009/10/28/accepted PY - 2009/11/26/entrez PY - 2009/11/26/pubmed PY - 2010/3/31/medline SP - 479 EP - 87 JF - Molecules and cells JO - Mol Cells VL - 28 IS - 5 N2 - 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 semiquantitative 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(2)O(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(2)O(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. SN - 0219-1032 UR - https://www.unboundmedicine.com/medline/citation/19936628/Expression_of_a_glutathione_reductase_from_Brassica_rapa_subsp__pekinensis_enhanced_cellular_redox_homeostasis_by_modulating_antioxidant_proteins_in_Escherichia_coli_ L2 - http://www.molcells.org/journal/view.html?year=2009&volume=28&number=5&spage=479 DB - PRIME DP - Unbound Medicine ER -