Expression, purification, and electrophysiological characterization of a recombinant, fluorescent Kir6.2 in mammalian cells.

Autor: Agasid MT; Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, United States., Wang X; Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, United States., Huang Y; Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, United States., Janczak CM; Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, United States., Bränström R; Department of Molecular Medicine and Surgery, Karolinksa Institutet, Stockholm, Sweden., Saavedra SS; Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, United States; BIO5 Institute, University of Arizona, Tucson, AZ 85721, United States. Electronic address: saavedra@email.arizona.edu., Aspinwall CA; Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, United States; BIO5 Institute, University of Arizona, Tucson, AZ 85721, United States; Department of Biomedical Engineering, University of Arizona, Tucson, AZ 85721, United States. Electronic address: aspinwal@email.arizona.edu.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2018 Jun; Vol. 146, pp. 61-68. Date of Electronic Publication: 2018 Feb 07.
DOI: 10.1016/j.pep.2018.01.015
Abstrakt: 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-Kir6.2Δ26. eGFP-Kir6.2Δ26 was expressed in HEK293 cells and a purification protocol developed. Electrophysiological characterization showed that eGFP-Kir6.2Δ26 retains native single channel conductance (64 ± 3.3 pS), mean open times (τ 1  = 0.72 ms, τ 2  = 15.3 ms) and ATP affinity (IC 50  = 115 ± 25 μM) when expressed in HEK293 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 Co 2+ -metal affinity chromatography and SEC was implemented for purification. Purified protein activity was assessed by reconstituting eGFP-Kir6.2Δ26 in black lipid membranes (BLMs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), l-α-phosphatidylinositol-4,5-bisphosphate (PIP 2 ) in a 89.5:10:0.5 mol ratio. Reconstituted eGFP-Kir6.2Δ26 displayed similar single channel conductance (61.8 ± 0.54 pS) compared to eGFP-Kir6.2Δ26 expressed in HEK293 membranes; however, channel mean open times increased (τ 1  = 7.9 ms, τ 2  = 61.9 ms) and ATP inhibition was significantly reduced for eGFP-Kir6.2Δ26 reconstituted into BLMs (IC 50  = 3.14 ± 0.4 mM). Overall, this protocol should be foundational for the production of purified Kir6.2 for future structural and biochemical studies.
(Copyright © 2018 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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