Cell-Free Membrane Protein Expression into Hybrid Lipid/Polymer Vesicles

Hybrid membranes comprised of diblock copolymers, and phospholipids have gained interest due to their unique properties that result from blending natural and synthetic components. The integration of membrane proteins into these synthetic membranes is an important step towards creating biomembrane systems for uses such as artificial cellular systems, biosensors, and drug delivery vehicles. Here, we outline a technique to create hybrid membranes composed of phospholipids and diblock copolymers. Next, we describe how membrane proteins can be co-translationally integrated into hybrid lipid/polymer membranes using a cell-free reaction. We then outline a method to monitor insertion and folding of a membrane-embedded channel protein into the hybrid membrane using a fluorescent-protein reporter and dye release assay, respectively. This method is expected to be applicable for a wide range of membrane proteins that do not require chaperones for co-translational integration into vesicles and provides a generalized protocol for expressing a membrane protein into a membrane mimetic.