Fig. 2
Biosynthesis and isolation of the recombinant Dmb hydrogenase subunits from E. coli. After IPTG induction of the recombinant genes, the bacteria were further cultivated overnight at 18oC; approx. MW – approximate, theoretically predicted molecular weight. (a) Biosynthesis of the large (LH) and small (SH) recombinant Dmb H2ase subunits in E. coli BL21(DE3)Gold. Lane M, protein molecular weight ladder, selected bands marked; lanes 1–2, E. coli BL21(DE3)Gold [SH_ pMCSG53]; lanes 3–4, E. coli BL21(DE3)Gold [LH U493M_ pMCSG53], lanes 5–6, E. coli BL21(DE3)Gold [LH U493C_ pMCSG53]; lane 7, E. coli BL21(DE3)Gold [SH_LH U493M_ pMCSG53]; lane 8, E. coli BL21(DE3)Gold [SH_LH U493C pMCSG53]. (b) Isolation and Immobilized metal affinity chromatography (IMAC) purification of the recombinant Dmb H2ase subunits from E. coli BL21(DE3)Gold. All the purified protein variants were obtained using the recombinant pMCSG53 constructs. Lane M, protein molecular weight ladder, selected bands marked; lane 1, purified LH U493M; lane 2, purified SH and truncated LH U493M – expression from the operon; lane 3, purified truncated LH U493C; lane 4, purified SH and truncated LH U493C – expression from the operon