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Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni 2+ ‐NTA affinity chromatography
Author(s) -
Mitchell David M.,
Gennis Robert B.
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00626-k
Subject(s) - rhodobacter sphaeroides , chemistry , cytochrome c oxidase , mutant , affinity chromatography , wild type , stereochemistry , biochemistry , chromatography , enzyme , photosynthesis , gene
A rapid and highly efficient method of purifying the aa 3 ‐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.