Formation of Cell-Sized Liposomes Incorporating a β‑Barrel-Structured Porin through Rehydration of a Phospholipid-Membrane Protein Dried Film

Giant unilamellar vesicles (GUVs) integrated with membrane proteins (proteo-GUVs) are attractive tools for visualizing membrane protein functions such as enzyme reactions and molecular transportation. In the dehydration–rehydration method, one of the methods used to form proteo-GUVs, they are formed by using a dried film containing phospholipids and membrane proteins through rehydration with an alternating current electric field and a supporting gel. However, these methods make it difficult to form proteo-GUVs under physiological salt concentration and charged phospholipid conditions or carry the risk of gel contamination of lipid membranes. Therefore, proteo-GUVs formed by these rehydration methods may be harmful to membrane proteins. Here, we propose a method for the formation of proteo-GUVs containing physiological salt concentrations and negatively charged phospholipids that do not require an electric field and a supporting gel. To investigate the molecular transport of modified outer membrane protein G (OmpG), OmpG-giant unilamellar vesicles (GUVs) and OmpG-large unilamellar vesicles (LUVs) were formed. The structure and function of different mutants reconstituted into LUVs were evaluated by using circular dichroism spectroscopy and electrophysiological measurements. In addition, the molecular transport of OmpG in GUVs was evaluated by monitoring the Ca²⁺ influx into GUVs and fluorescent molecule leakage from GUVs through OmpG nanopores. We found that the amount of Ca²⁺ influx into GUVs through the OmpG nanopores depended on the pore size of OmpG. Our method for forming proteo-GUVs can be applied for the functional evaluation of β-barrel porin and in biological sensors using β-barrel porin.