Expression、Purification And Activity Assay Of A New Thermostable Superoxide Dismutase Gene

The Thermostable Superoxide Dismutase gene which we study is from the new strain of Geobacillus. The length of ORF is 1236 bp which encodes a protein of 411 amino acid residues. And the molecular weight of this SOD is 47.25 kD. By bioinformatics analysis, we found at the 366-373 bp of this SOD is a conserved sequence DVWEHAYY which also contains in SOD protein from other species. Via predicting its tertiary structure, we found this SOD gene has nine a helixes and three β folded structure, the shape of which is like fence. To study the function of Thermostable Superoxide Dismutase, we coloned the ORF of this SOD gene into fusion expression plasmid pET-28a(+), then transformed this recombinant plasmid to E.coli BL21(DE3) for expression under induction of IPTG. Then, we identified the expression product by SDS-PAGE. The molecular weight of this fusion protein is 48.07 kD. The expressed protein was simply purified by the process of His-tag affinity chromatography. We found that the recombinant protein is still active at 90℃. Treat the recombinant protein in different temperature, can remove most of the other protein without affecting the activity of the recombinant protein and the final purity can reach 70%. In addition, we also constructed the recombinant pFastBac HTB-SOD, then transferred it to Eukaryotic expression systetm(Bac to Bac system). We identified the recombinant plasmid Bacmid-SOD by PCR. Then we applied the technology of lipofection to transfer the recombinant Bacmid-SOD into BmN, the infected cells were collected and cultured for 5 generations, then collected recombinant baculovirus. At last, we used WST-1 method for determine the SOD activity of the products expressed in both prokaryotic and eukaryotic. Through Comparing the specific activity of thermostable SOD expressed in the two different systems, we established the way of fermentating the thermostable SOD by using prokaryotic expression system. At last, we marked the fermented Thermostable SOD protein with Cy5 Fluorescent dye, then observed the distribution of SOD in BALB/c mice under the vivo imaging system. The results showed that the thermostable SOD protein was enriched in the kidney of mouse and no significant enrichment in heart、spleen and other organs. After one hour of administration, it still enriched in kidney. The mechanism of this phenomenon needs to be further study.