Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni(2+)-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 Ni(2+)-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.

journal_name

FEBS Lett

journal_title

FEBS letters

authors

Mitchell DM,Gennis RB

doi

10.1016/0014-5793(95)00626-k

subject

Has Abstract

pub_date

1995-07-10 00:00:00

pages

148-50

issue

1

eissn

0014-5793

issn

1873-3468

pii

0014-5793(95)00626-K

journal_volume

368

pub_type

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