Expression and purification of recombinant human inward rectifier K+ (KCNJ) channels in Saccharomyces cerevisiae

被引：19

作者：

D'Avanzo, Nazzareno ^{[1
,2
,3
]}

Cheng, Wayland W. L. ^{[2
,3
]}

Xia, Xiaobing ^{[1
]}

Dong, Liang ^{[1
]}

Savitsky, Pavel ^{[1
]}

Nichols, Colin G. ^{[2
,3
]}

Doyle, Declan A. ^{[1
]}

机构：

[1] Univ Oxford, Struct Genom Consortium, Oxford OX3 7DQ, England

[2] Washington Univ, Sch Med, Dept Cell Biol & Physiol, St Louis, MO 63110 USA

[3] Washington Univ, Sch Med, Ctr Invest Membrane Excitabil Dis, St Louis, MO 63110 USA

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2010年 / 71卷 / 01期

基金：

英国惠康基金;

关键词：

KCNJ; Inward rectifier; Expression; Purification; Ion channels; Saccharomyces cerevisiae; POTASSIUM CHANNEL; CRYSTAL-STRUCTURE; MECHANISM; PROTEINS; SUBUNITS; FAMILY;

D O I：

10.1016/j.pep.2010.01.010

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The inward rectifier family of potassium (KCNJ) channels regulate vital cellular processes including cell volume, electrical excitability, and insulin secretion. Dysfunction of different isoforms have been linked to numerous diseases including Bartter's, Andersen-Tawil, Smith-Magenis Syndromes, Type II diabetes mellitus, and epilepsy, making them important targets for therapeutic intervention. Using a family-based approach, we succeeded in expressing 10 of 11 human KCNJ channels tested in Saccharomyces cerevisiae. GFP-fusion proteins showed that these channels traffic correctly to the plasma-membrane suggesting that the protein is functional. A 2-step purification process can be used to purify the KCNJ channels to-dispersed form. After incorporation into liposomes, Rb-86(+) flux assays confirm >95% purity in a mono the functionality of the purified proteins as inward rectifier potassium channels. (C) 2010 Elsevier Inc. Ail rights reserved.

引用

页码：115 / 121

页数：7

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