Abstract: Staphylococcal enterotoxin M (SEM) is a secretory superantigen encoded by the νSa genomic islands of Staphylococcus aureus. In this study, the sem gene from S. aureus without N-terminal signal peptide was subcloned into the prokaryotic expression vector pET-28a (+) to construct the recombinant plasmid pET-28a (+)-ΔNspsem and the recombinant expression plasmid was then transformed into E. coli Rosetta (DE3) competent cells. The positive clones, induced by IPTG, effectively expressed His-tag containing soluble ΔNspSEM fusion protein in E. coli Rosetta (DE3). The expression conditions including expression vector, time, IPTG concentration and temperature were optimized. Purified His-ΔNspSEM fusion protein was obtained by Ni2+-Sepharose affinity chromatography. Mass spectrometric analysis indicated that the amino acid sequence of the fusion protein was 96.3% similar to that of SEM. Circular dichroism revealed ΔNspSEM, whose 6 × His sequence was cleaved by thrombin, was rich in β-sheet (35%) and β-turn (21%) but low in α-helix (16%). The fluorescence emission spectrum of ΔNspSEM exhibited identical tryptophan emission peak (341 nm) with excitation at 278 and 295 nm. The time-dependent thermal stability of ΔNspSEM obtained at 100 ℃ by SDS-PAGE indicated that the recombinant protein had relatively high thermal stability. To conclude, our results showed that the ΔNspSEM recombinant protein was successfully expressed and that the purified protein exhibited a compact conformation similar to the natural one in solution, which can provide a basis for insight into the structure and function of SEM protein.

Key words: staphylococcal enterotoxin M, prokaryotic expression, purification, mass spectrometry, circular dichroism, fluorescence emission spectroscopy, thermal stability