کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8359500 | 1542295 | 2018 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression, purification, and electrophysiological characterization of a recombinant, fluorescent Kir6.2 in mammalian cells
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Expression, purification, and electrophysiological characterization of a recombinant, fluorescent Kir6.2 in mammalian cells Expression, purification, and electrophysiological characterization of a recombinant, fluorescent Kir6.2 in mammalian cells](/preview/png/8359500.png)
چکیده انگلیسی
The inwardly rectifying K+ (Kir) channel, Kir6.2, plays critical roles in physiological processes in the brain, heart, and pancreas. Although Kir6.2 has been extensively studied in numerous expression systems, a comprehensive description of an expression and purification protocol has not been reported. We expressed and characterized a recombinant Kir6.2, with an N-terminal decahistidine tag, enhanced green fluorescent protein (eGFP) and deletion of C-terminal 26 amino acids, in succession, denoted eGFP-Kir6.2Î26. eGFP-Kir6.2Î26 was expressed in HEK293â¯cells and a purification protocol developed. Electrophysiological characterization showed that eGFP-Kir6.2Î26 retains native single channel conductance (64â¯Â±â¯3.3â¯pS), mean open times (Ï1â¯=â¯0.72â¯ms, Ï2â¯=â¯15.3â¯ms) and ATP affinity (IC50â¯=â¯115â¯Â±â¯25â¯Î¼M) when expressed in HEK293â¯cells. Detergent screening using size exclusion chromatography (SEC) identified Fos-choline-14 (FC-14) as the most suitable surfactant for protein solubilization, as evidenced by maintenance of the native tetrameric structure in SDS-PAGE and western blot analysis. A two-step scheme using Co2+-metal affinity chromatography and SEC was implemented for purification. Purified protein activity was assessed by reconstituting eGFP-Kir6.2Î26 in black lipid membranes (BLMs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1â²-rac-glycerol) (POPG), l-α-phosphatidylinositol-4,5-bisphosphate (PIP2) in a 89.5:10:0.5â¯mol ratio. Reconstituted eGFP-Kir6.2Î26 displayed similar single channel conductance (61.8â¯Â±â¯0.54â¯pS) compared to eGFP-Kir6.2Î26 expressed in HEK293 membranes; however, channel mean open times increased (Ï1â¯=â¯7.9â¯ms, Ï2â¯=â¯61.9â¯ms) and ATP inhibition was significantly reduced for eGFP-Kir6.2Î26 reconstituted into BLMs (IC50â¯=â¯3.14â¯Â±â¯0.4â¯mM). Overall, this protocol should be foundational for the production of purified Kir6.2 for future structural and biochemical studies.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 146, June 2018, Pages 61-68
Journal: Protein Expression and Purification - Volume 146, June 2018, Pages 61-68
نویسندگان
Mark T. Agasid, Xuemin Wang, Yiding Huang, Colleen M. Janczak, Robert Bränström, S. Scott Saavedra, Craig A. Aspinwall,