Expression, purification and spectroscopic studies of full-length Kir3.1 channel C-terminus.
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.
Leach
Robert N
RN
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. r.n.leach@leeds.ac.uk
Boyett
Mark R
MR
Findlay
John B C
JB
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D015870:Gene Expression; D006639:Histidine; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D024661:Potassium Channels, Inwardly Rectifying; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; D006639:Histidine