Giant Protein Vesicles for Studying Membrane Proteins

Abstract

Giant vesicles offer a great potential as a native like cell-mimetic environment for protein reconstitutions and subsequent protein characterization. However, protein reconstitution into giant vesicles is not straightforward and the assembly procedures of giant vesicles generally preclude protein reconstitution. This has so far limited the use of these vesicles as a general biomimetic platform for studying membrane proteins and in novel membrane protein-based biotechnological applications.In this study we present a method for formation of giant protein vesicles (GPV). The method supports formation of GPV in physiological ionic strength buffers such as phosphate buffered saline, and does not require specialized equipment, specialized lipids and peptides or a dehydration/rehydration step. Moreover, the amount of reconstituted protein in the GPV may be controlled by the lipid-to-protein ratio of the large vesicles. We characterized GPV formation using the spinach aquaporin SoPIP2;1 and E. coli aquaporin AqpZ as model proteins of α-helical polytopic membrane proteins. Functional protein reconstitution into GPV is demonstrated with the light-driven proton pump Bacteriorhodopsin.We suggest that the described method may constitute a general and versatile method for the formation of GPV. This may open up for the possibilities of a more general use of membrane proteins in biotechnological applications including membrane protein biosensors, drug discovery, bioreactors, nano-machines and novel separation technologies.