Virus-Like Particles (VLPs) as a Platform for Hierarchical Compartmentalization.

Hierarchically self-assembled structures are common in biology, but it is often challenging to design and fabricate synthetic analogs. The archetypal cell is defined by hierarchically organized multicompartmentalized structures with boundaries that delineate the interior from exterior environments and is an inspiration for complex functional materials. Here, we have demonstrated an approach to the design and construction of a nested protein cage system that can additionally incorporate the packing of other functional macromolecules and exhibit some of the features of a minimal synthetic cell-like material. We have demonstrated a strategy for controlled co-packaging of subcompartments, ferritin (Fn) cages, together with active cellobiose-hydrolyzing β-glycosidase enzyme macromolecules, CelB, inside the sequestered volume of the bacteriophage P22 capsid. Using controlled in vitro assembly, we were able to modulate the stoichiometry of Fn cages and CelB encapsulated inside the P22 to control the degree of compartmentalization. The co-encapsulated enzyme CelB showed catalytic activity even when packaged at high total macromolecular concentrations comparable to an intracellular environment. This approach could be used as a model to create synthetic protein-based protocells that can confine smaller functionalized proto-organelles and additional macromolecules to support a range of biochemical reactions.