PubMed 14644043

Referenced in Channelpedia wiki pages of: none

Automatically associated channels: Kir3.1

Title: Expression, purification and spectroscopic studies of full-length Kir3.1 channel C-terminus.

Authors: Robert N Leach, Mark R Boyett, John B C Findlay

Journal, date & volume: Biochim. Biophys. Acta, 2003 Dec 1 , 1652, 83-90

PubMed link:

A polypeptide corresponding to the full-length C-terminal cytoplasmic domain of a G-protein-regulated inwardly rectifying potassium channel (Kir3.1) bearing a hexahistidine (His6) tag was produced by DNA recombinant overexpression techniques in Escherichia coli. This permitted the isolation of approximately 5 mg of pure protein per liter of bacterial culture. Further purification by size exclusion chromatography (SEC) of the C-terminal domain revealed that it exists predominantly as a dimer. The secondary structure was estimated using circular dichroism measurements that indicated the presence of approximately 35% beta-sheet and approximately 15% alpha-helix. G-protein betagamma subunits incubated with His-tagged Kir3.1 C-terminal domain, bound to immobilized metal affinity chromatography (IMAC) resin, copurified with the peak of specifically eluted recombinant protein. These observations demonstrate that full-length Kir3.1 C-terminus can be purified in a stable conformation capable of binding proteins known to activate Kir3 channels and may contain elements involved in channel assembly.