Insertion of Functional Proteins into Bilayer Lipid Membrane using a Cell-Free Expression System

Abstract

The incorporation of well-conformed membrane proteins within lipid bilayer is an important challenge because these proteins play a major role in every living cell and are key factors in cell-cell interaction, signal transduction and transport of ions and nutrients. To insert an integral membrane protein in a lipid bilayer it is important to separate the lipid bilayer from the supporting solid substrate in order to minimize interactions of the protein with the substrate and to provide adequate space for the protein incorporation. We chose to produce two kind of lipid bilayer membrane, a suspended membrane in which alpha hemolysin nanopores were produced and incorporated and a tethered Bilayer Lipid Membrane (tBLM), spaced from the surface by a tethering molecule as a polyethylene glycol (PEG), for the incorporation of a transmembrane protein like Aquaporin Z. The two membrane proteins were produced directly on the top of the lipid bilayers using a cell-free expression system, without any purification. This alternative technique is not affected by cell physiology and allows producing membrane proteins in a correct conformation without toxicity limitation, protein aggregation or misfolding. To demonstrate that these proteins produced with this cell free expression system are inserted and functional in a lipid bilayer, we used Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), Atomic Force Microscopy (AFM), Surface Plasmon Resonance (SPR) and ion current recording experimentations.