Expression and One-Step Purification of a Fully Active Polyhistidine-Tagged Cytochromebc1Complex fromRhodobacter sphaeroides

ThefbcBandfbcCgenes encoding cytochromesbandc1of thebc1complex were extended with a segment to encode a polyhistidine tag linked to their C-terminal sequence allowing a one-step affinity purification of the complex. Constructions were madein vitroin a pUC-derived background using PCR amplification. The modifiedfbcoperons were transferred to a pRK derivative plasmid, and this was used to transform thefbc−strain ofRhodobacter sphaeroides,BC17. The transformants showed normal rates of growth. Chromatophores prepared from these cells showed kinetics of turnover of thebc1complex on flash activation which were essentially the same as those from wild-type strains, and analysis of the cytochrome complement and spectral and thermodynamic properties by redox potentiometry showed no marked difference from the wild type. Chromatophores were solubilized and mixed with Ni-NTA–Sepharose resin. A modification of the standard elution protocol in which histidine replaced imidazole increased the activity 20-fold. Imidazole modified the redox properties of hemec1, suggesting ligand displacement and inactivation when this reagent is used at high concentration. The purified enzyme contained all four subunits in an active dimeric complex. This construction provides a facile method for preparation of wild-type or mutantbc1complex, for spectroscopy and structural studies.