Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni2+ NTA
affinity chromatography.
David M. Mitchell & Robert B. GennisFEBS Letters
368 (1995)148 150
SUMMARY: 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 Ni2+ 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 wild type 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.