Engineering a Self-Assembled Protein Cage for Targeted Dual Functionalization.

Self-assembled protein cages are attractive scaffolds for organizing various proteins of interest (POIs) toward applications in synthetic biology and medical science. However, specifically attaching multiple POIs to a single protein cage remains challenging, resulting in diversity among the functionalized particles. Here, we present the engineering of a self-assembled protein cage, Mi3, capable of independently recruiting two different POIs using SpyCatcher (SC)/SpyTag (ST) and DogCatcher (DC)/DogTag (DT) chemistries, thereby reducing variability between assemblies. Using fluorescent proteins as models, we demonstrate controlled targeting of two different POIs onto Mi3 protein cages both in vitro and inside living cells. Furthermore, dual functionalization of the Mi3 protein cage with a membrane-targeting peptide and β-galactosidase resulted in the construction of membrane-bound enzyme assemblies in , leading to a 69.6% enhancement in substrate utilization across the membrane. This versatile protein cage platform provides dual functional nanotools for biological and biomedical applications.