Nowadays, recombinant therapeutic proteins have been widely produced and consumed. For the safety and effectiveness of the protein production, an auto-inducible expression vector is required to replace inducer interference, which is uneconomic and could be harmful. In this research, an auto-inducible expression plasmid, pCAD2_sod (a pBR322 derivate plasmid), which was under dps (RpoS-dependent gene) promoter control, was modified to provide RpoS at earlier phase. Hence, accumulates more target protein and resulting a new plasmid, pCAD2+_sod. pCAD2_sod had been constructed to automatically induces the expression of recombinant superoxide dismutase (SOD) from Staphylococcus equorum (rMnSODSeq) in the stationary growth phase of Escherichia coli. This work aimed to obtain pCAD2+_sod and determine the expression level of rMnSODSeq on mRNA and protein level. A synthetic rpoS coding region under rpoD promoter control (prpoD_rpoS) was inserted to pCAD2_sod and generated pCAD2+_sod. The rMnSODSeq (24.3 kDa) produced from pCAD2+_sod was ~ 1.5 fold higher at 37 °C and more intense at 43 °C compared to that from pCAD2_sod, likewise shifted to earlier phase (after 1 h of incubation), as shown in the SDS-PAGE. The dismutase activity was also retained after zymography assay. The mRNA level from pCAD2+_sod was determined by qPCR and gave quantification cycle (Cq) values of cDNA lowest among others. It made the relative quantification (RQ) of the mRNA expression towards rho reference gene were high. The prpoD_rpoS insertion shifts and increases the rMnSODSeq production from stationary to exponential phase. The pCAD2+_sod plasmid is potential for further recombinant protein productions.
A recombinant hybrid of manganese dependent-superoxide dismutase of Staphylococcus equorum and S. saprophyticus has successfully been overexpressed in Escherichia coli BL21(DE3), purified, and characterized. The recombinant enzyme suffered from degradation and aggregation upon storage at −20 °C, but not at room temperature nor in cold. Chromatographic analysis in a size exclusion column suggested the occurrence of dimeric form, which has been reported to contribute in maintaining the stability of the enzyme. Effect of monovalent (Na+, K+), divalent (Ca2+, Mg2+), multivalent (Mn2+/4+, Zn2+/4+) cations and anions (Cl−, SO4 2−) to the enzyme stability or dimeric state depended on type of cation or anion, its concentration, and pH. However, tremendous effect was observed with 50 mM ZnSO4, in which thermostability of both the dimer and monomer was increased. Similar situation was not observed with MnSO4, and its presence was detrimental at 200 mM. Finally, chelating agent appeared to destabilize the dimer around neutral pH and dissociate it at basic pH. The monomer remained stable upon addition of ethylene diamine tetraacetic acid. Here we reported unique characteristics and stability of manganese dependent-superoxide dismutase from S. equorum/saprophyticus.