Modular interior loading and exterior decoration of a virus-like particle.

Virus-like particles (VLPs) derived from the bacteriophage P22 offer an interesting and malleable platform for encapsulation and multivalent presentation of cargo molecules. The packaging of cargo in P22 VLP is typically achieved through genetically enabled directed in vivo encapsulation. However, this approach does not allow control over the packing density and composition of the encapsulated cargos. Here, we have adopted an in vitro assembly approach to gain control over cargo packaging in P22. The packaging was controlled by closely regulating the stoichiometric ratio of cargo-fused-scaffold protein and wild-type scaffold protein during the in vitro assembly. In a "one-pot assembly reaction" coat protein subunits were incubated with varied ratios of wild-type scaffold protein and cargo-fused-scaffold protein, which resulted in the encapsulation of both components in a co-assembled capsid. These experiments demonstrate that an input stoichiometry can be used to achieve controlled packaging of multiple cargos within the VLP. The porous nature of P22 allows the escape and re-entry of wild-type scaffold protein from the assembled capsid but scaffold protein fused to a protein-cargo cannot traverse the capsid shell due to the size of the cargo. This has allowed us to control and alter the packing density by selectively releasing wild-type scaffold protein from the co-assembled capsids. We have demonstrated these concepts in the P22 system using an encapsulated streptavidin protein and have shown its highly selective interaction with biotin or biotin derivatives. Additionally, this system can be used to encapsulate small molecules coupled to biotin, or display large proteins, that cannot enter the capsid and thus remain available for the multivalent display on the exterior of the capsid when attached to a flexible biotinylated linker. Thus, we have developed a P22 system with controlled protein cargo composition and packing density, to which both small and large molecules can be attached at high copy number on the interior or exterior of the capsid.