(167e) Multi-Compartmentalized, Giant Protein Vesicles for Artificial Cell Application

Living cells have several compartmentalized organelles that perform specific functions. Such multiple compartments enable spatial confinements of incompatible enzymes and substrates, separating each other and allowing each reaction to occur in an optimized space. Hence, multi-compartmentalization is one of the essential structural properties of living cells to recapitulate in artificial cell research. Synthetic giant vesicles with a size range larger than 10μm have been utilized in artificial cell research as a powerful tool to study the structure and functions of biological cell membranes, such as lateral heterogeneities, membrane budding and fusion. Among diverse types of synthetic vesicles, globular protein vesicles (GPVs) formed through self-assembly of recombinant fusion proteins have been highlighted as a potential platform to create artificial cells, facilitating the incorporation of multiple proteins with controlled amounts and orientations. However, the formation of GPVs by self-assembly limits vesicle size control within a few hundred nm to a few µm, leading to difficulties in the practical engineering of GPVs toward multi-compartmentalized artificial cell applications. Herein, we utilize the thin-film rehydration method, which is a widely used method to construct giant lipid vesicles and polymer vesicles, to build giant GPVs with multi-compartment structure. Giant GPVs are formed by hydration of fusion protein thin films fabricated on substrates by evaporating solvent of the protein solution followed by swelling. This fabrication method enables us to obtain the target GPV size range between 10 µm and 100 µm, a typical size range of eukaryotic cells. By leveraging the swelling process of thin films, as a proof-of-concept, we encapsulate diverse types of cargo inside giant GPVs, including smaller sized, self-assembled vesicles, protein coacervates and fibrils, and model synthetic polymers and hydrogels. These internal compartments and synthetic polymers are utilized as models of artificial organelles and cytosol. Giant GPVs, which include multiple compartments and cell-like composition within the vesicle, would serve as a promising model of artificial cells due to their cell-like structure and composition.