Expression And Purification Of Recombinant Human Fusion Enzyme SOD2/3 In E.Coli And Its Pharmacological Activity Assay In Vitro

Superoxide dismutase (SOD) is a kind of metal enzymes that can accelerate superoxide anion (O2-) disproportionation reaction. There are three kinds of SOD in human and other mammals: intracellular Cu, Zn-SOD (SOD1), Mn-SOD (SOD2) in mitochondria and extracellular EC-SOD (SOD3) containing Cu and Zn. Especially, Cu, Zn-SOD is widely used in radiation protection, anti-aging, cancer prevention and control, etc..However, the half-life of Cu, Zn-SOD and Mn-SOD in vivo is short, and can not bind with the endothelial cell specifically which greatly limits their potential applications. Extracellular superoxide dismutase (SOD3) is able to located at the cell surface or heparin-rich extracellular matrix take advantage of its C terminal heparin-binding domain. Its half-life is longer in the body(about 10h), which just enough make up for the shortcomings of Cu, Zn-SOD and Mn-SOD.in addition, SOD3 can also regulate the extent of NO. It is a better drug than SOD1 and SOD2. Bcause so far, recombinant expression of SOD3 is still poor in both prokaryotes and yeast, it is important to find/construct new SOD analogs with high SOD activity, long half-life of SOD3 and cell targeting abilities.In this study, the genes encoding SOD2 and C terminal heparin-binding domain of SOD3 were fused together and cloned into pET28a(+). The recombinant plasmid MnSOD-ECSOD3-pET28a was transformed into E. coli BL21 (DE3) where the fusion SOD2/3 was expressed. When induced by final concentration of 0.5mM IPTG and 2.5mM Mn2+ at 15℃for 15-20 hrs, the amount of recombinant hybrid enzyme was 30%～40% that of the total bacterial protein. Recombinant SOD2/3 was successfully purified by Ni chalting affinity chromatography, heparin affinity chromatography and DEAE-52 ion-exchange chromatography; purified hybrid showed inhibiting effect on the growth of Hela cells in vitro.