Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni2+‐NTA affinity chromatography
- 10 July 1995
- journal article
- Published by Wiley in FEBS Letters
- Vol. 368 (1) , 148-150
- https://doi.org/10.1016/0014-5793(95)00626-k
Abstract
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.Keywords
This publication has 22 references indexed in Scilit:
- The binuclear CuA centre of cytochrome oxidaseBiochimica et Biophysica Acta (BBA) - Bioenergetics, 1994
- Vibrational Characteristics of Mutant and Wild-type Carbon Monoxy Cytochrome c Oxidase: Evidence for a Linear Arrangement of Heme a, a3, and CuBJournal of the American Chemical Society, 1994
- The CuAsite of the caa3‐type oxidase of Bacillus subtilis is a mixed‐valence binuclear copper centreFEBS Letters, 1994
- Internal Electron Transfer in Cytochrome c Oxidase Is Coupled to the Protonation of a Group Close to the Bimetallic SiteBiochemistry, 1994
- A Loop between Transmembrane Helixes IX and X of Subunit I of Cytochrome c Oxidase Caps the Heme a-Heme a3-CuB CenterBiochemistry, 1994
- Binuclear centre structure of terminal protonmotive oxidasesFEBS Letters, 1993
- Intramolecular electron transfer in cytochrome c oxidase: a cascade of equilibriaBiochemistry, 1992
- Cloning, sequencing and deletion from the chromosome of the gene encoding subunit I of the aa3‐type cytochrome c oxidase of Rhodobacter sphaeroidesMolecular Microbiology, 1992
- A general method for rapid site-directed mutagenesis using the polymerase chain reactionGene, 1990
- Sequence diversity among related genes for recognition of specific targets in DNA moleculesJournal of Molecular Biology, 1983