Purification and partial characterization of recombinant Cu, Zn containing superoxide dismutase of Cordyceps militaris in E.coli.
The cDNA of Cu, Zn containing superoxide dismutase from the Cordyceps militaris SH (cm-SOD) was overexpressed in Escherichia coli BL 21 (DE3) using the pET-21a expression vector. The recombinant cell overexpressed the protein corresponding to 35+/-3% of total bacterial protein in cytosol. The purification was performed through three steps: DEAE-FF, CM-52, and G-100. After this purification procedure, a specific activity of 27272.7 U/mg of protein was reached, corresponding to 6.1-fold purification with a yield of 85.0%. The purity was homogeneous by SDS-PAGE analysis and 94.2+/-1.0% by CZE analysis. A subunit molecular mass of the recombinant enzyme was 15704 Da with a Cu and Zn element. In addition, the dimeric and polymeric structures were observed on MALDI-TOF-MS. Isoelectric point value of 7.0 was obtained for the recombinant enzyme that was sensitive to H2O2 and KCN. The recombinant enzyme remained 80+/-2% residual activity at pH 7.8, at 50 degrees C for 4h incubation. The properties: N-terminal amino acid sequence (the first 12 amino acid residues), pI, subunit molecular mass, thermo-stability of the purified recombinant SOD are similar to that of the native Cu, Zn-SOD from C. militaris (N-cm-SOD).
Wang
Zunsheng
Z
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, P.O. Box 365, Shanghai 200237, PR China. wangzsy@yahoo.com.cn
He
Zhuojing
Z
Shen
Qiong
Q
Gu
Yuxiang
Y
Li
Suxia
S
Yuan
Qinsheng
Q
D000595:Amino Acid Sequence; D032661:Cordyceps / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008969:Molecular Sequence Data; D011190:Potassium Cyanide / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000302:isolation & purification*
D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D011190:Potassium Cyanide