Size-controlled synthesis of phase separated protein condensates with interfacial protein cages

Phase separation of specific proteins into liquidic condensates is a key mechanism to form membrane-less organelles, which organize diverse cellular processes in space and time. These protein condensates hold immense potential as biomaterials that can contain specific sets of biomolecules with extremely high densities and dynamic liquid properties. Despite their appeal, methods to manipulate protein condensate materials remain largely unexplored. Here, we developed a one-pot assembly method to synthesize coalescence-free protein condensates from a few μm to 100 nm sizes with surface-stabilizing protein cages. We discovered that large protein cages (~30 nm), with precisely tuned interaction strengths to condensates, could effectively localize on condensate surfaces and block coalescence during phase separation. This approach proved applicable to diverse condensates with varying compositions and fluidities. Condensate sizes were concisely controlled by modulating condensate/cage ratios. In addition, we successfully visualized the 3D structures of intact protein condensates with interfacial cages with cryo-electron tomography (ET). Protein cages formed monolayer shells on protein condensates, where cages were slightly buried in condensates with contact angles lower than 90 degree. These cage-covered protein condensates maintained dynamic properties, including the capacity for selective material exchange or recruitment from the external environment.