FEBS Letters | |
Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni2+‐NTA affinity chromatography | |
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
【 预 览 】
Files | Size | Format | View |
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RO201912020301307ZK.pdf | 335KB | download |