Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni2+‐NTA affinity chromatography

FEBS Letters

Gennis, Robert B.¹  Mitchell, David M.¹ 

[1] School of Chemical Sciences, University of Illinois, Urbana, IL 61801, USA

关键词: Cytochrome c oxidase;    Ni-chelate chromatography;    Membrane protein;    Bioenergetics;    Rhodobacter sphaeroides;    NTA;    nitrilotriacetic acid;    SDS-PAGE;    sodium dodecyl sulfate-polyacrylamide gel electrophoresis;    EPR;    electron paramagnetic resonance;    UV/VIS;    ultraviolet/visible;    PCR;    polymerase chain reaction;    ICP-AES;    inductively-coupled plasma-atomic emission spectroscopy;   

DOI  :  10.1016/0014-5793(95)00626-K

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 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 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.

【 授权许可】

Unknown