Membrane Protein Synthesis and Reconstitution into Monodisperse Giant Unilamellar Vesicles Produced by Microfluidics

Giant unilamellar vesicles (GUVs) have been used as artificial cell membrane models composed of a lipid bilayer, and various preparation methods were proposed so far. Recently, fabrication methods of GUVs with uniform size using microfluidic channels have been reported from several groups including our laboratory [1]–[3]. In this study, we tested direct insertion by in situ synthesis of membrane proteins into the membranes of monodisperse GUVs generated using our microfluidic system. Here, two membrane proteins were synthesized by an in vitro transcription-translation (IVTT) system from the protein-encoded DNA to be directly inserted into the GUV membrane. First, we synthesized the nanopore-forming membrane protein α-HL inside or outside of GUVs and evaluated its function to permeate only small molecules by free diffusion. Second, we synthesized the bacterial multidrug transporter EmrE outside of GUVs and investigated its insertion into the membrane. Our results indicate that a variety of membrane proteins can be reconstituted into monodisperse GUVs with the present protocol.