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.
D'Avanzo
Nazzareno
N
Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK.
Cheng
Wayland W L
WW
Xia
Xiaobing
X
Dong
Liang
L
Savitsky
Pavel
P
Nichols
Colin G
CG
Doyle
Declan A
DA
D001671:Biochemistry / Q000379:methods*; D003001:Cloning, Molecular; D006801:Humans; D024661:Potassium Channels, Inwardly Rectifying / Q000302:isolation & purification* / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins