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, DT Mi3 ST , 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 DT Mi3 ST protein cages both in vitro and inside living cells. Furthermore, dual functionalization of the DT Mi3 ST protein cage with a membrane-targeting peptide and β-galactosidase resulted in the construction of membrane-bound enzyme assemblies in Escherichia coli , 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.