Protocells with Fluorescent Response to Extracellular Signals

This protocol describes the generation of protocells mimicking the signal transduction of living cells using giant unilamellar liposomes generated by the phase transfer method. This method allows the efficient encapsulation of cell-free protein synthesis reagents and plasmids encoding enzyme switches comprising an affinity tag, transmembrane domain, and β-glucuronidase mutant. Antibody binding to affinity tags on the protocell surface induces oligomerization and activation of the enzyme inside the protocells, producing a fluorescent signal from a fluorogenic substrate. The detailed procedure includes emulsion generation, liposome formation, protein synthesis inside the liposomes, and fluorescence detection. The protocells generated using this protocol provide a well-controlled system for studying signal transduction across membranes and the manipulation of physiological processes originating from living cells.