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

Authors
Publisher
Elsevier B.V.
Publication Date
Volume
1652
Issue
2
Identifiers
DOI: 10.1016/j.bbapap.2003.07.001
Keywords
  • Kir3.1
  • Circular Dichroism Spectroscopy
  • Secondary Structure
  • Protein Expression
Disciplines
  • Biology

Abstract

Abstract 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 ∼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 ∼35% β-sheet and ∼15% α-helix. G-protein βγ 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.

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