Expression, purification and spectroscopic studies of full-length Kir3.1 channel C-terminus
Expression, purification and spectroscopic studies of full-length Kir3.1 channel C-terminus
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DOI：
10.1016/j.bbapap.2003.07.001
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发表时间：
2003-12-01
影响因子：
3.2
通讯作者：
Findlay, JBC
中科院分区：
文献类型：
--
作者：
Leach, RN;Boyett, MR;Findlay, JBC
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 similar to 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 similar to 35% beta-sheet and similar to 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. (C) 2003 Elsevier B.V. All rights reserved.
