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

Expression and purification of recombinant human inward rectifier K+ (KCNJ) channels in Saccharomyces cerevisiae
Expression and purification of recombinant human inward rectifier K+ (KCNJ) channels in Saccharomyces cerevisiae
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, (86)Rb(+) flux assays confirm the functionality of the purified proteins as inward rectifier potassium channels.
KCNJ, inward rectifier, expression, purification, ion channels, saccharomyces cerevisiae
1046-5928
115-121
D'Avanzo, Nazzareno
0af1c4a7-ffe0-44d9-90cc-caa53591750f
Cheng, Wayland W.L.
160e9e4c-b341-4da9-b4f2-3be8d456dd3e
Xia, Xiaobing
ad07038c-becb-451e-b53c-ee5c4c5fbde4
Dong, Liang
c8314915-7a9a-49ab-b91e-0bdf36a5d6bd
Savitsky, Pavel
e2a33817-3762-414c-bf7e-68673955ede5
Nichols, Colin G.
042f1de5-f614-4b02-83fb-0b56b7f62e4d
Doyle, Declan A.
f85f52c8-ce43-4f15-bd06-1df106f73b26
D'Avanzo, Nazzareno
0af1c4a7-ffe0-44d9-90cc-caa53591750f
Cheng, Wayland W.L.
160e9e4c-b341-4da9-b4f2-3be8d456dd3e
Xia, Xiaobing
ad07038c-becb-451e-b53c-ee5c4c5fbde4
Dong, Liang
c8314915-7a9a-49ab-b91e-0bdf36a5d6bd
Savitsky, Pavel
e2a33817-3762-414c-bf7e-68673955ede5
Nichols, Colin G.
042f1de5-f614-4b02-83fb-0b56b7f62e4d
Doyle, Declan A.
f85f52c8-ce43-4f15-bd06-1df106f73b26

D'Avanzo, Nazzareno, Cheng, Wayland W.L., Xia, Xiaobing, Dong, Liang, Savitsky, Pavel, Nichols, Colin G. and Doyle, Declan A. (2010) Expression and purification of recombinant human inward rectifier K+ (KCNJ) channels in Saccharomyces cerevisiae. Protein Expression and Purification, 71 (1), 115-121. (doi:10.1016/j.pep.2010.01.010). (PMID:20064617)

Record type: Article

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, (86)Rb(+) flux assays confirm the functionality of the purified proteins as inward rectifier potassium channels.

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e-pub ahead of print date: 11 January 2010
Published date: May 2010
Keywords: KCNJ, inward rectifier, expression, purification, ion channels, saccharomyces cerevisiae
Organisations: Centre for Biological Sciences

Identifiers

Local EPrints ID: 342294
URI: http://eprints.soton.ac.uk/id/eprint/342294
ISSN: 1046-5928
PURE UUID: 2a8bdc7a-1fd7-403b-8bf1-a2a83779c179

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Date deposited: 20 Aug 2012 14:22
Last modified: 02 Dec 2019 20:56

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