Recombinant Fusion Proteins with Embedded Sensing\nFunctions as Versatile Tools for Protocell Development

Sensory capabilities are crucial\nfor cells to interact with their\nenvironment. To mimic these functions in synthetic cells, we developed\nsensory globular protein vesicles (GPVs) made entirely of recombinant\nfusion proteins through self-assembly under aqueous conditions. GPVs\ndemonstrate sensory functions via the formation of the FKBP-FRB ternary\ncomplex with the signaling molecule, rapamycin. The sensory domain\nof FKBP or FRB was genetically fused to a fluorescent protein and\nleucine zipper, which self-assemble into vesicles by forming amphiphilic\nbuilding blocks through high-affinity binding to a counter leucine\nzipper fused to an elastin-like polypeptide (ELP) above its lower\ncritical solution temperature. We observed intervesicle aggregation\nin a time- and concentration-dependent manner upon rapamycin binding,\nconfirmed by colocalization studies and statistical analysis. This\nsystem enhances our understanding of protein vesicle functionality\nfor sensing and offers a basis for exploring GPVs as models to replicate\nkey cellular processes in synthetic cells.