Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni(2+)-NTA affinity chromatography.
A rapid and highly efficient method of purifying the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been developed. This method relies upon a six-histidine affinity tag fused to the C-terminus of subunit I, which confers to the oxidase a high affinity for Ni(2+)-nitrilotriacetic acid (NTA) agarose. The histidine-tagged oxidase can be purified rapidly and with high yield by one affinity chromatography step, starting with solubilized membranes. The purified oxidase is > 95% pure and possesses structural and functional characteristics of the wildtype enzyme. The six-histidine tag can be easily added to pre-constructed site-directed mutants of subunit I, increasing the availability of purified cytochrome c oxidase mutants for biophysical and biochemical studies.
Mitchell
D M
DM
School of Chemical Sciences, University of Illinois, Urbana 61801, USA.
Gennis
R B
RB
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D009838:Oligodeoxyribonucleotides; D009942:Organometallic Compounds; D012242:Rhodobacter sphaeroides / Q000201:enzymology*
D009838:Oligodeoxyribonucleotides; D009942:Organometallic Compounds; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D003576:Electron Transport Complex IV; D009571:Nitrilotriacetic Acid