Overexpression of a eukaryotic glutathione reductase gene from Brassica campestris improved resistance to oxidative stress in Escherichia coli.
Glutathione reductase (GR) plays an essential role in a cell's defense against reactive oxygen metabolites by sustaining the reduced status of an important antioxidant glutathione. We constructed a recombinant plasmid based on the expression vector pET-18a that overexpresses a eukaryotic GR from Brassica campestris (BcGR) in Escherichia coli. For comparative analyses, E. coli GR (EcGR) was also subcloned in the same manner. The transformed E. coli with the recombinant constructs accumulated a high level of GR transcripts upon IPTG induction. Also, Western blot analysis showed overproduction of the BcGR protein in a soluble fraction of the transformed E. coli extract. When treated with oxidative stress generating reagents such as paraquat, salicylic acid, and cadmium, the BcGR overproducing E. coli exhibited a higher level of growth and survival rate than the control E. coli strain, but it was not as high as the E. coli strain transformed with the inducible EcGR. The translated amino acid sequences of BcGR and EcGR share 37.3% identity but all the functionally known important residues are conserved. It appears that eukaryotic BcGR functions in a prokaryotic system by providing protection against oxidative damages in E. coli.
Yoon
Ho-Sung
HS
Institute of Agricultural Science and Technology, Kyungpook National University, Daegu 702-701, Republic of Korea.
Lee
In-Ae
IA
Lee
Hyoshin
H
Lee
Byung-Hyun
BH
Jo
Jinki
J
D017123:Bacteriophage T7 / Q000235:genetics; D001937:Brassica / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D016373:DNA, Antisense / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005980:Glutathione Reductase / Q000235:genetics* / Q000378:metabolism; D030781:Organisms, Genetically Modified / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D011401:Promoter Regions, Genetic; D013997:Time Factors
D016373:DNA, Antisense; D005980:Glutathione Reductase