Prokaryotic Expression And Funcational Characterization Of Superoxidase Dismutase From Cristaria Plicata

The freshwater mussel Cristaria plicata, which is of great economical importance and known as "pearl bivalves" in the aquaculture industry of China, has been suffering serious problems due to the outbreak of diseases.Thus, understanding of the immunity of freshwater mussel is crucial for diseases management and development of sustainable mussel culture and pearl production.Superoxide dismutases (SODs) are one family of important antioxidant metalloenzymes involved in scavenging the high level of reactive oxygen species (ROS) to protect cells. The present study aims to clone the full-length icCuZnSOD cDNA from the freshwater mussel C. plicata, to express recombinant Cp-icCuZnSOD (rCpSOD) enzyme in Escherichia coli and purify it, to characterize the enzyme activity and stability of rCpSOD protein in vitro, and to evaluate protective effects of rCpSOD.In the present study, the intracellular CuZnSOD gene of C. plicata (Cp-icCuZnSOD) has been cloned using the rapid amplification of cDNA ends (RACE) technique and RT-PCR.The DNA products and vector pET-30a were digested by the Kpn I,EcoR I enmyzes, and then ligated.The recombinant plasmids were transformed into the prokaryotic cell Rosetta-gami(DE3)and induced with IPTG, supplemented with and without Cu2+Zn2+at 20℃or 37℃, and then inclusion body and soluble forms of rCpSOD protein were purified by using the native Ni2+ affinity chromatography and examined the specific activity respectively. The stability of rCpSOD with the highest specific activity to temperature, pH and denaturant was examed. The antigenic specificities of fusion proteins were analyzed by Western-blot with mouse antiserum against rCpSOD. Finally, alcohol-injured cell-damaging model was established to discusse rCpSOD enzmye could protect hepatocyte L02 cells from oxidative damage or not.SDS-PAGE result showed that rCp-icCuZnSOD induced at 37℃was almost aggregated to form inclusion bodies, soluble rCp-icCuZnSOD was obtained when induced at 20℃, and the expression level was enhanced when induced at 20℃supplemented with Cu2+/Zn2+. The result of bioactivity assay showed that the SOD enzyme activity of inclusion bodies induced at 37℃was lower than that was induced at 20℃, the specific activity was lower 1000 U/mg supplemented with Cu2+/Zn2+or not.The SOD activity of soluble rCpSOD induced at 20℃was significantly enhanced, the specific activity was the highest with 5300 U/mg induced with Cu2+/Zn2+. This current study shows that low temperature and supplement with Cu2+/Zn2+are beneficial to soluble expression level and bioactivity enhancement of rCpSOD. The enzyme stability assay shew that thepurified rCp-icCuZnSOD enzyme maintained more than 80% activity at temperature up to 60℃, at pH 2.0-9.0, and was resistant to 8 mol·L-1 urea or 8% SDS. In addition, the addition of active rCpSOD enzmye could protect hepatocyte L02 cells from oxidative damage as assessed using an alcohol-injured human liver cell model. Mouse polyclonal antisera were developed against the purified rCpSOD protein, and the fusion proteins were recognized by mouse antiserum using Western-blot. In addition, the addition of active rCpSOD enzmye could protect hepatocyte L02 cells from oxidative damage as assessed using an alcohol-injured human liver cell model.