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

David M. Mitchell; Robert B. Gennis

doi:10.1016/0014-5793(95)00626-K

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

David M. Mitchell, Robert B. Gennis

Biochemistry

Research output: Contribution to journal › Article › peer-review

Abstract

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 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.

Original language	English (US)
Pages (from-to)	148-150
Number of pages	3
Journal	FEBS Letters
Volume	368
Issue number	1
DOIs	https://doi.org/10.1016/0014-5793(95)00626-K
State	Published - Jul 10 1995

Keywords

Bioenergetics
Cytochrome c oxidase
Membrane protein
Ni-chelate chromatography
Rhodobacter sphaeroides

ASJC Scopus subject areas

Biophysics
Structural Biology
Biochemistry
Molecular Biology
Genetics
Cell Biology

Online availability

10.1016/0014-5793(95)00626-K

Library availability

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Cite this

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title = "Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni2+-NTA affinity chromatography",

abstract = "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 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.",

keywords = "Bioenergetics, Cytochrome c oxidase, Membrane protein, Ni-chelate chromatography, Rhodobacter sphaeroides",

author = "Mitchell, {David M.} and Gennis, {Robert B.}",

note = "Funding Information: Acknowledgements.{"} This work was supported by a grant from the National Institutes of Health (R.B.G.): HL16101.",

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T1 - Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni2+-NTA affinity chromatography

AU - Mitchell, David M.

AU - Gennis, Robert B.

N1 - Funding Information: Acknowledgements." This work was supported by a grant from the National Institutes of Health (R.B.G.): HL16101.

PY - 1995/7/10

Y1 - 1995/7/10

N2 - 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 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.

AB - 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 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.

KW - Bioenergetics

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KW - Membrane protein

KW - Ni-chelate chromatography

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