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

Nazzareno D'Avanzo; Wayland W.L. Cheng; Xiaobing Xia; Liang Dong; Pavel Savitsky; Colin G. Nichols; Declan A. Doyle

doi:10.1016/j.pep.2010.01.010

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

Nazzareno D'Avanzo, Wayland W.L. Cheng, Xiaobing Xia, Liang Dong, Pavel Savitsky, Colin G. Nichols, Declan A. Doyle

Research output: Contribution to journal › Article › peer-review

18 Scopus citations

Abstract

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 >95% purity in a mono-dispersed form. After incorporation into liposomes, ⁸⁶Rb⁺ flux assays confirm the functionality of the purified proteins as inward rectifier potassium channels.

Original language	English
Pages (from-to)	115-121
Number of pages	7
Journal	Protein Expression and Purification
Volume	71
Issue number	1
DOIs	https://doi.org/10.1016/j.pep.2010.01.010
State	Published - May 2010

Keywords

Expression
Inward rectifier
Ion channels
KCNJ
Purification
Saccharomyces cerevisiae

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

abstract = "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 >95% purity in a mono-dispersed form. After incorporation into liposomes, 86Rb+ flux assays confirm the functionality of the purified proteins as inward rectifier potassium channels.",

keywords = "Expression, Inward rectifier, Ion channels, KCNJ, Purification, Saccharomyces cerevisiae",

author = "Nazzareno D'Avanzo and Cheng, {Wayland W.L.} and Xiaobing Xia and Liang Dong and Pavel Savitsky and Nichols, {Colin G.} and Doyle, {Declan A.}",

note = "Funding Information: This work was supported by NIH Grant HL54171 (to CGN) and the Structural Genomics Consortium . The Structural Genomics Consortium is a registered charity (No. 1097737) funded by the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck and Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust.",

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AU - Dong, Liang

AU - Savitsky, Pavel

AU - Nichols, Colin G.

AU - Doyle, Declan A.

N1 - Funding Information: This work was supported by NIH Grant HL54171 (to CGN) and the Structural Genomics Consortium . The Structural Genomics Consortium is a registered charity (No. 1097737) funded by the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck and Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust.

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

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