Characterization of fish Cu/Zn-superoxide dismutase and its protection from oxidative stress.
Copper/zinc superoxide dismutase was cloned from the zebrafish ( Danio rerio). The full coding region of the zebrafish superoxide dismutase (ZSOD) complementary DNA was ligated with pET-20b(+) and successfully expressed in Escherichia coli strain AD494(DE3)pLysS. The active enzyme was purified by His tagging. The ZSOD yield was 6 mg from 0.2 L of E. coli culture, and the specific activity was 2000 U/mg as assayed using a RANSOD kit. The enzyme stability was characterized by reaction to temperature, pH, and detergent treatment. The results showed enzyme activity was still active after heat treatment at 70 degrees C for 10 minutes, resistant to pH treatment from 2.3 to 12, and resistant to treatment with sodium dodecyl sulfate (SDS) under 4%. In addition, the recombinant ZSOD was used to protect fish from 100 ppm of paraquat-induced oxidative injury by soaking fish larva in 55 micro g/ml SOD enzyme. The results were significant.
Ken
Chuian-Fu
CF
Graduate Institute of Life Sciences, National Defense Medical Center, Taiwan.
Lin
Chi-Tsai
CT
Shaw
Jei-Fu
JF
Wu
Jen-Leih
JL
D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006540:Herbicides / Q000494:pharmacology; D007814:Larva / Q000187:drug effects / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D010269:Paraquat / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D018076:DNA, Complementary; D006540:Herbicides; D029961:Zebrafish Proteins; D013482:Superoxide Dismutase; D010269:Paraquat