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

Journal article

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.

Authors: D'Avanzo, N; Cheng, W; Xia, X; Dong, L; Savitsky, P

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Elsevier
• Journal: Protein Expression and Purification
• Volume: 71
• Issue: 1
• Pages: 115-121
• Publication date: 2010-01-01
• DOI: 10.1016/j.pep.2010.01.010
• EISSN: 1096-0279
• ISSN: 1046-5928
• Language: English
• Subjects: Recombinant Proteins; isolation and purification; Potassium Channels, Inwardly Rectifying; Cloning, Molecular; metabolism; methods; Saccharomyces cerevisiae; Subcellular Fractions; Protein Transport