Expression and one-step purification of a fully active polyhistidine-tagged cytochrome bc1 complex from Rhodobacter sphaeroides.
The fbcB and fbcC genes encoding cytochromes b and c1 of the bc1 complex 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 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 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 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
M
Department of Biochemistry, University of Illinois, Urbana, Illinois, 61801, USA.
Salcedo-Hernandez
R
R
Bechmann
G
G
Kuras
R
R
Gennis
R B
RB
Crofts
A R
AR
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics; D003575:Cytochromes c1 / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers; D014450:Electron Transport Complex III / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006639:Histidine; D008969:Molecular Sequence Data; D010455:Peptides; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics
D003573:Cytochrome b Group; D017931:DNA Primers; D010455:Peptides; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D003575:Cytochromes c1; D014450:Electron Transport Complex III