RAPID PURIFICATION OF WILDTYPE AND MUTANT CYTOCHROME-C-OXIDASE FROM RHODOBACTER-SPHAEROIDES BY NI2+-NTA AFFINITY-CHROMATOGRAPHY

被引：149

作者：

MITCHELL, DM ^{[1
]}

GENNIS, RB ^{[1
]}

机构：

[1] UNIV ILLINOIS,SCH CHEM SCI,URBANA,IL 61801

来源：

FEBS LETTERS | 1995年 / 368卷 / 01期

关键词：

CYTOCHROME C OXIDASE; NI-CHELATE CHROMATOGRAPHY; MEMBRANE PROTEIN; BIOENERGETICS; RHODOBACTER SPHAEROIDES;

D O I：

10.1016/0014-5793(95)00626-K

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

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.

引用

页码：148 / 150

页数：3

共 25 条

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