Cloning of active human manganese superoxide dismutase and its oxidative protection in Escherichia coli.
Superoxide radical (O2*-) is a toxic byproduct of oxidative metabolism that extensively damages cellular macromolecules and organelles. Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide (H2O2) and molecular oxygen (O2) thus providing a biological defense against oxygen toxicity. The structural gene of human manganese superoxide dismutase (hMnSOD) was successfully cloned into the pET46Ek/LIC by using a Ligation Independent Cloning (LIC) technique. The recombinant human MnSOD was expressed in E. coli strain BL21(DE3)pLysS and purified to homogeneity by Ni2+ -NTA. Supplementation of Mn2+ in the bacterial growth media was proven to be crucial for production of enzymatically active hMnSOD. The recombinant enzyme revealed a specific activity up to 2,857 U mg(-1) as measured by inhibition of photoreduction of nitroblue tetrazolium (NBT). The molecular weight of each subunit was estimated to be 22 kDa by SDS-PAGE. More interestingly, E. coli expressing hMnSOD provides resistance against oxidative stress induced by the herbicide paraquat up to 1.2 mM. These findings gain insights into the biochemical characterization and significant roles of oxidative-protection of the hMnSOD in biological systems.
Yainoy
Sakda
S
Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University 2 Prannok Rd, Bangkoknoi, Bangkok 10700, Thailand.
Isarankura-Na-Ayudhya
Chartchalerm
C
Tantimongcolwat
Tanawut
T
Prachayasittikul
Virapong
V
D000595:Amino Acid Sequence; D000975:Antioxidants / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D016166:Free Radical Scavengers / Q000378:metabolism*; D006540:Herbicides / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010269:Paraquat / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000975:Antioxidants; D016166:Free Radical Scavengers; D006540:Herbicides; D011994:Recombinant Proteins; D013482:Superoxide Dismutase; D010269:Paraquat