It made the relative quantification (RQ) of the mRNA expression towards rho reference gene were high. The mRNA level from pCAD 2 +_ sod was determined by qPCR and gave quantification cycle (Cq) values of cDNA lowest among others. The dismutase activity was also retained after zymography assay. The rMnSODSeq (24.3 kDa) produced from pCAD 2 +_ sod was ~ 1.5 fold higher at 37 ☌ and more intense at 43 ☌ compared to that from pCAD 2_ sod, likewise shifted to earlier phase (after 1 h of incubation), as shown in the SDS-PAGE. Method and resultsĪ synthetic rpoS coding region under rpoD promoter control (p rpoD_rpoS) was inserted to pCAD 2 _sod and generated pCAD 2 +_ sod. This work aimed to obtain pCAD 2 +_ sod and determine the expression level of rMnSODSeq on mRNA and protein level. pCAD 2 _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. Hence, accumulates more target protein and resulting a new plasmid, pCAD 2 +_ sod. In this research, an auto-inducible expression plasmid, pCAD 2 _sod (a pBR322 derivate plasmid), which was under dps (RpoS-dependent gene) promoter control, was modified to provide RpoS at earlier phase. 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. Nowadays, recombinant therapeutic proteins have been widely produced and consumed.
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