High-Throughput Screening of Membrane Transport Proteins in Synthetic Lipid Vesicles

Membrane transport proteins mediate the exchange of ions, small molecules, or macromolecules across the cell membrane and play critical roles in many biological systems. Engineered transporters have myriad applications that include biosensing, metabolic engineering, and experimental tools for probing neural function. Nevertheless, transporter engineering has been limited by the lack of robust and quantitative high-throughput assays suitable for screening large transporter libraries. Cell-based assays are difficult due to the toxicity of heterologously expressed membrane proteins, the confounding effects of endogenous transporters, and the inability to precisely control the intracellular environment. We present a new method for high-throughput transporter screening in synthetic lipid vesicles. Microfluidic flow focusing produces double emulsion templates that, through a novel dewetting mechanism, are converted into monodisperse giant unilamellar vesicles. The vesicles can be loaded with cell-free expression systems to express the desired transporters in the membrane, where they function. This allows the transporter variants to be assayed and sorted at high throughput using commercial fluorescence activated cell sorters. Synthetic lipid vesicles enable high-throughput screening of membrane transport proteins, and represent a robust platform for the directed evolution of transporters with new biochemical properties.