|ZFIN ID: ZDB-PUB-070625-9|
Replacement of Buried Cysteine from Zebrafish Cu/Zn Superoxide Dismutase and Enhancement of Its Stability via Site-Directed Mutagenesis
Ken, C.F., Lin, C.T., Wen, Y.D., and Wu, J.L.
|Source:||Marine biotechnology (New York, N.Y.) 9(3): 335-342 (Journal)|
|Registered Authors:||Wu, Jen-Leih|
|Keywords:||Cu/Zn superoxide dismutase, I-Mutant 2.0, oxidative stress, site-direct mutagenesis, thermostability|
|PubMed:||17549562 Full text @ Mar. Biotechnol.|
Ken, C.F., Lin, C.T., Wen, Y.D., and Wu, J.L. (2007) Replacement of Buried Cysteine from Zebrafish Cu/Zn Superoxide Dismutase and Enhancement of Its Stability via Site-Directed Mutagenesis. Marine biotechnology (New York, N.Y.). 9(3):335-342.
ABSTRACTZebrafish Cu/Zn-superoxide dismutase (ZSOD1) has one free cysteine (Cys-7) in a first beta-strand with lower thermostability. We predicted the stability would be increased with single-point mutation at 70 degrees C via the I-Mutant 2.0 server, and generated a mutant SOD with replacement of the free Cys to Ala (ZSODC7A) by site-directed mutagenesis. The mutant was expressed and purified from the Escherichia coli strain AD494(DE3)pLysS and the yield was 2 mg from 0.4 L of culture. The ZSODC7A was heated at 90 degrees C. In a time-dependent assay, the time interval for 50% inactivation was 32 min, and its thermal inactivation rate constant K (d) was 2 x 10(-2) min(-1). The mutant was still activated in broad pH range (2.3-12), and had only a moderate effect under sodium dodecyl sulfate treatment. The calculated specific activity of the mutant was 3980 U/mg, twice that of wild-type ZSOD1. In addition, we soaked fish larva with equal enzyme units of either ZSOD1 or ZSODC7A for 2 h, and then stressed them with 100 ppm of paraquat to induce oxidative injury. The survival rate was significant.
- Genes / Markers (1)