(28v) Designing Recombinant Protein-Based Sensory Vesicle System

Sensing is a vital function in living cells to communicate with each other as well as allow controlled release of molecules from the vesicle membrane in response to the stimuli. Developing synthetic cells using protein building blocks capable of sensing continues to be challenging as traditional building blocks do not offer such advanced biological functions that are mediated by proteins. However, recombinant protein-based self-assembled vesicles open new opportunities to incorporate functionally folded, sensory proteins into membranes with a high modularity. Here, we develop self-assembled protein vesicles that can sense and respond to specific signaling molecules by leveraging the ternary complex system involving FKBP-Rapamycin-FRB. The sensory protein domain, either FRB- or FKBP-, was genetically fused to a fluorescent protein-leucine zipper fusion proteins (i.e., FRB-mCherry-Z_E or FKBP-GFP-Z_E). These sensory-fluorescent-leucine zipper fusion proteins self-assemble into vesicles with counter leucine zipper fused with elastin-like polypeptide (Z_R-ELP) by forming amphiphilic protein building blocks in water at the temperature above the lower critical solution temperature of ELP. We have successfully created vesicles self-assembled from FRB-mCherry-Z_E and FKBP-GFP-Z_E with Z_R-ELP. Rapamycin-induced interaction of these vesicles was characterized using dynamic light scattering, fluorescence and confocal microscopy techniques, and fluorescence resonance energy transfer (FRET) assays. This would pave way for further exploration of various applications of potential protein vesicles to recapitulate minimal cellular functions.