A convenient protocol for generating giant unilamellar vesicles containing SNARE proteins using electroformation

Abstract

Reconstitution of membrane proteins in artificial membranes is an essential prerequisite for functional studies that depend on the context of an intact membrane. While straight-forward protocols for reconstituting proteins in small unilamellar vesicles were developed many years ago, it is much more difficult to prepare large membranes containing membrane proteins at biologically relevant concentrations. Giant unilamellar vesicles (GUVs) represent a model system that is characterised by low curvature, controllable tension, and large surface that can be easily visualised with microscopy, but protein insertion is notoriously difficult. Here we describe a convenient method for efficient generation of GUVs containing functionally active SNARE proteins that govern exocytosis of synaptic vesicles. Preparation of proteo-GUVs requires a simple, in-house-built device, standard and inexpensive electronic equipment, and employs a straight-forward protocol that largely avoids damage of the proteins. The procedure allows upscaling and multiplexing, thus providing a platform for establishing and optimizing preparation of GUVs containing membrane proteins for a diverse array of applications.

Self‐Assembly of Giant Unilamellar Vesicles by Film Hydration Methodologies

“…A) Reproduced under the terms and conditions of the Creative Commons Attribution License (CC BY) . Copyright 2017, The Authors; B) Reproduced under the terms and conditions of the Creative Commons Attribution 4.0 International License . Copyright 2018, The Authors, published by Springer Nature; C) Homemade device; D) Reproduced under the terms and conditions of the Creative Commons Attribution (CC‐BY) License .…”

“…Besides the setup, the conditions can vary between different studies without any obvious reasons ( Table 1 ). In terms of frequency, regardless of ITO/Pt electrodes or lipid/polymer amphiphiles, most studies uses 10 Hz, which is sometimes followed by a decrease of the frequency to 4–5 Hz, said to be required for GUV detachment . Some experiments also use frequencies as low as 1 Hz or as high as 500 Hz for lipids .…”

Differential diffusional properties in loose and tight docking prior to membrane fusion

“…Protein expression and purification. Rattus norvegicus-derived SNARE proteins (syntaxin-1A (183-288) (39), SNAP-25 (cysteine free) (40), synaptobrevin-2 (wildtype (WT) (41), Δ84 (42), and I45A,M46A (AA) (17,43)), 1-96 (44), and synaptobrevin-2 fragment (49-96) (20) were expressed and purified as described before (17,45). In short, proteins were expressed in Escherichia coli strain BL21 (DE3) and purified via nickel-nitrilotriacetic acid affinity chromatography (Qiagen) followed by ion exchange chromatography on an Äkta system (GE Healthcare).…”

“…It consists of syntaxin, SNAP-25, and synaptobrevin fragment 49-96. The complex was obtained by mixing the monomers overnight at 4°C, followed by purification of the complex using ion exchange chromatography (MonoQ column) in a buffer containing CHAPS as described before (20,45).…”

“…GUV preparation. Giant unilamellar vesicles were prepared from small proteoliposomes containing ΔN complex by electroformation using in-house-built electroformation chambers containing platinum wires (46) using a protocol de-scribed previously (45).…”