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

David M. Mitchell, Robert B. Gennis

Research output: Contribution to journalArticlepeer-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 languageEnglish (US)
Pages (from-to)148-150
Number of pages3
JournalFEBS Letters
Volume368
Issue number1
DOIs
StatePublished - 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

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