How to Tailor Giant Proteoliposomes

In this project we address the challenge of incorporation of transmembrane proteins with defined orientation in biomimetic giant unilamellar vesicles (GUVs). The reconstitution of biologically relevant transmembrane proteins, like receptors or channel proteins, into GUVs makes them easily accessible to biophysical investigation using patch-clamp or optical microscopy while preserving their natural configuration. Our strategy combines two approaches: cell-free eukaryotic protein expression and biomimetic model systems. The cell-free protein expression system is based on insect lysates [1]. It provides endoplasmic reticulum (ER)-based vesicles which enable signal-induced translocation and post-translational modification, e.g. glycosylation and phosphorylation. Further the expressed protein is inserted into the ER membrane corresponding to the natural process in a living cell.Starting from small ER vesicles of approximately one micrometer diameter we applied electro-swelling to achieve giant proteoliposomes [2]. Our recent work showed that the efficiency of this method can be improved substantially by the presence of synthetic lipids in the electro-swelling process. As an example, we introduced the one-transmembrane protein heparin-binding epidermal growth factor-like factor Hb-EGF-eYFP in GUV membranes aided by the lipid DOPC. We applied single-molecule fluorescence microscopy to detect and localize the protein, and characterized their mobility in the mixed natural-synthetic membrane. In addition, we introduced biotinylated lipids that enabled us to immobilize the protein-decorated GUVs to streptavidin coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.[1] Kubick et al. Current Topics in Membranes 09.[2] Shaklee et al. ChemBioChem 10.