Abstract
Functional studies of membrane-bound channels, transporters or signal transducers require that the protein of interest resides in a membrane that separates two compartments. One approach that is commonly used to prepare these systems is to reconstitute the protein in liposomes. An intermediate step of this method is purification of the protein, which typically involves solubilization of the native membrane using detergent. The use of detergents often results in removal of lipids surrounding the protein, which may alter its structure and function. Here, we have employed a method for isolation of membrane proteins with a disc of their native lipids to develop an approach that allows transfer of the purified membrane protein to liposomes without the use of any detergents.
Highlights
Removal of membrane proteins from their native membrane environment can destabilize these proteins and/ or alter their functional properties[1,2]
It was added to the mitochondrial preparation, non-solubilized membranes were removed at 1.1 × 105 g (40 min), and the styrene maleic acid (SMA)-cytochrome c oxidase (CytcO) native nanodiscs were isolated using affinity chromatography (HisTrap HP, GE healthcare) as described previously[11]
The average diameter of the SMA-CytcO-liposomes was found to be ~110 nm, determined using Tunable Resistive Pulse Sensing (TRPS): the liposome sample was diluted 200 times in a buffer composed of 20 mM HEPES, 200 mM KCl and applied on top of a stretchable pore (NP200)
Summary
S. cerevisiae strain MOY764 with a C-terminal His7Strep tag on Cox1311 and a strain with a C-terminal His[10] tag on Cox[622] were used. It was added to the mitochondrial preparation, non-solubilized membranes were removed at 1.1 × 105 g (40 min), and the SMA-CytcO native nanodiscs were isolated using affinity chromatography (HisTrap HP, GE healthcare) as described previously[11]. We developed two different methods to prepare liposomes and to incorporate the SMA-CytcO nanodiscs, using sonication or extrusion. After extrusion the liposomes were kept for 20 min at room temperature, at 4 °C for 1 hour and on ice. The average diameter of the SMA-CytcO-liposomes was found to be ~110 nm, determined using Tunable Resistive Pulse Sensing (TRPS) (qNano, Izon Science, UK): the liposome sample was diluted 200 times (to 1–1.5 nM CytcO) in a buffer composed of 20 mM HEPES, 200 mM KCl and applied on top of a stretchable pore (NP200). The measurement was calibrated with carboxylated polystyrene beads CPC100B (ModeDia 110 nm, Izon)
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