Expression and One-Step Purification of a Fully Active Poly-Histidine Tagged Cytochrome bc1-Complex from Rhodobacter sphaeroides.

Mariana Guergova-Kuras§, Ruben Salcedo-Hernandez#, Georg Bechmann, Richard Kuras, Robert B. Gennis# and Antony R. Crofts#§ Departments of Biochemistry# and Microbiology, and Center for Biophysics and Computational Biology§, University of Illinois, Urbana, IL 61801

ABSTRACT.

The fbcB and fbcC genes encoding cytochromes b and c1 of the bc1-complex, were extended with a segment to encode a poly-histidine tag linked to their C-terminal sequence allowing a one-step affinity purification of the complex. Constructions were made in vitro in a pUC-derived background using PCR amplification. The modified fbc operons were transferred to a pRK-derivative plasmid, and this was used to transform the fbc- strain of Rhodobacter sphaeroides, BC17. The transformants showed normal rates of growth. Chromatophores prepared from these cells showed kinetics of turnover of the bc1-complex on flash activation which were essentially the same as those from wild-type strains, and analysis of the cytochrome complement, 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 heme c1, 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 mutant bc1-complex, for spectroscopy and structural studies.


Guergova-Kuras, M., Salcedo-Hernandez, R., Bechmann, G., Kuras, R., Gennis, R.B. and Crofts, A.R. (1998) Expression and one-step purification of a fully active poly-histidine tagged bc1 complex from Rhodobacter sphaeroides. Protein Expression and Purification 15, 370-380.