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.
Highlights
Giant unilamellar vesicles (GUVs) represent artificial vesicles with a diameter of >1 μm
Electroformation of GUVs can be performed by applying an alternating electric field in the formation chamber that either consists of two glasses coated with conductive material that are separated by a spacer, or that contains two Pt electrodes
Additional parameters have a strong influence on GUV formation such as chamber volume, chamber cleanliness and deterioration due to repeated use, concentration of lipids, the method of lipid drying, and the composition of the buffer used for electroformation
Summary
Giant unilamellar vesicles (GUVs) represent artificial vesicles with a diameter of >1 μm. Preparation of protein-containing GUVs (proteo-GUVs) has been a challenging task due to the fact that GUV-formation usually includes steps that can denature proteins Such steps may include: (i) formation of a lipid mixture from lipids diluted in organic solvents, (ii) drying, and (iii) formation of vesicles in a salt-free environment (but note that there are ongoing efforts to optimize protocols utilizing physiological ionic strength buffers, see e.g. ref.[13]). The preparation of proteo-GUVs still requires time-consuming optimization as protocols are frequently difficult to reproduce between laboratories (own experience and personal communication with other researchers in the field), largely because not all variables affecting the outcome are controlled and optimised This includes, for instance, design of the electroformation chamber including slide resistance when using ITO slides, spacer thickness (that is necessary for calculating the electric field), and for Pt electrodes information about the thickness and the axial distance between the wires. The parameters of the applied electric field are critical for the outcome, with the results depending on the chamber geometry and the precise voltage-time profile of the applied electric field
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