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

✍ Scribed by Robert N. Leach; Mark R. Boyett; John B.C. Findlay

• Book ID: 104003235
• Publisher: Elsevier Science
• Year: 2003
• Tongue: English
• Weight: 479 KB
• Volume: 1652
• Category: Article
• ISSN: 1570-9639
• DOI: 10.1016/j.bbapap.2003.07.001

✦ Synopsis

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 f 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 f 35% h-sheet and f 15% a-helix. G-protein hg subunits incubated with His-tagged Kir3.1 Cterminal 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.