Expression, Purification, and Electrophysiological Characterization of a Recombinant, Fluorescent Kir6.2 in Mammalian Cells

Overview

Journal Protein Expr Purif

Specialty Molecular Biology

Date 2018 Feb 8

PMID 29409958

Citations 2

Authors

Mark T Agasid

Xuemin Wang

Yiding Huang

Colleen M Janczak

Robert Branstrom

S Scott Saavedra

Craig A Aspinwall

Affiliations

Soon will be listed here.

Abstract

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 = 0.72 ms, τ = 15.3 ms) and ATP affinity (IC = 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-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) 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 = 7.9 ms, τ = 61.9 ms) and ATP inhibition was significantly reduced for eGFP-Kir6.2Δ26 reconstituted into BLMs (IC = 3.14 ± 0.4 mM). Overall, this protocol should be foundational for the production of purified Kir6.2 for future structural and biochemical studies.

Citing Articles

Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K channels.

Heitz B, Branstrom R, Yang W, Huang Y, Moede T, Leibiger I Sci Rep. 2021; 11(1):21539.

PMID: 34728728 PMC: 8564548. DOI: 10.1038/s41598-021-00988-y.

Importance of the Choice of a Recombinant System to Produce Large Amounts of Functional Membrane Protein hERG.

Vasseur L, Cens T, Wagner R, Saint N, Kugler V, Chavanieu A Int J Mol Sci. 2019; 20(13).

PMID: 31261773 PMC: 6651182. DOI: 10.3390/ijms20133181.

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