Expression and oxidative stress tolerance studies of glutaredoxin from cyanobacterium Synechocystis sp. PCC 6803 in Escherichia coli.
Glutaredoxin (Grx), which has been found widely in bacteria, plant, and mammalian cells, is an electron carrier for ribonucleotide reductase and a general glutathione-disulfide reductase of importance for redox regulation. The open reading frame designated ssr2061 from cyanobacterium Synechocystis sp. PCC 6803 was found as a homologous gene coding for Grx. The amino acid sequence deduced from ssr2061 shares high identity with that of Grxs from other organisms. In the present study, the protein of Grx2061 encoded by ssr2061 was successfully overexpressed as soluble fraction in Escherichia coli BL21 (DE3). The recombinant protein was purified to near homogenity by two steps involving immobilized metal affinity chromatography and gel filtration chromatography with a yield of 22% and a specific activity of 41.5 micromol NADPH oxidized per milligram of protein in the 2-hydroxyethyl disulfide assay. The pET-2061 transformed Escherichia coli cells showed higher Grx activity and tolerance to H(2)O(2) mediated growth inhibition compared to cells transformed with the vector alone. This suggests that overexpression of Grx from Synechocystis sp. PCC 6803 may provide protection to E. coli cells against oxidative stress mediated by H(2)O(2).
Li
Min
M
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, PR China.
Huang
Wei
W
Yang
Qing
Q
Liu
Xingguo
X
Wu
Qingyu
Q
D000222:Adaptation, Physiological; D000595:Amino Acid Sequence / Q000235:genetics; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D015870:Gene Expression / Q000235:genetics*; D054477:Glutaredoxins; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008969:Molecular Sequence Data; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D046939:Synechocystis / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic
D004220:Disulfides; D054477:Glutaredoxins; D016877:Oxidants; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D010088:Oxidoreductases