Reconstitution of proteins on electroformed giant unilamellar vesicles

In vitro reconstitution of simplified biological systems from molecular parts has proven to be a powerful method for investigating the biochemical and biophysical principles underlying cellular processes. In recent years, there has been a growing interest in reconstitution of protein-membrane interactions to understand the critical role played by membranes in organizing molecular-scale events into micron-scale patterns and protrusions. However, while all reconstitution experiments depend on identifying and isolating an essential set of soluble biomolecules, such as proteins, DNA, and RNA, reconstitution of membrane-based processes involves the additional challenge of forming and working with lipid bilayer membranes with composition, fluidity, and mechanical properties appropriate for the process at hand. Here we discuss a selection of methods for forming synthetic lipid bilayer membranes and present a versatile electroformation protocol that our lab uses for reconstituting proteins on giant unilamellar vesicles. This synthetic membrane-based approach to reconstitution offers the ability to study protein organization and activity at membranes under more cell-like conditions, addressing a central challenge to accomplishing the grand goal of "building the cell."