Heterologous expression and characterization of a Superoxide Dismutase from the archeon Thermoplasma acidophilum

Enzymes derived from extremophiles, in particular from thermophilic and hyperthermophilic archaeas have industrial relevance because of their high stability towards heat, pressure, detergents and solvents in comparison to their bacterial homologs. Superoxide dismutases (SODs) are enzymes that catalyze the dismutation of superoxide to produce molecular oxygen and hydrogen peroxide, constituting an effective defense mechanism for the cell against harmful levels of superoxide. Thermoplasma acidophilum is a thermoacidophilic archaeon that grows at 59°C and pH 1.0-2.0 and the genome contains an open reading frame (ORF) encoding a SOD: Ta0013 (NCBI accession number: NP393491). Prediction analysis was performed to position Ta0013 into the Fe-SOD families using the SODa server. The gene was amplified and cloned into the expression vector pET-28a(+) to add a 6xHis-tag at the C-terminal end region. The recombinant protein was expressed in E. coli BL21-pLysS and purified by Ni-NTA affinity chromatography. The protein has a molecular mass of 25 kDa. Native electrophoresis was performed to confirm a tetrameric complex. SOD activity was assessed in the presence of KCN or H₂O₂ through zymograms. Our results confirm a functional tetrameric Fe-SOD. We present a detailed enzymatic characterization of the enzyme.