High‐Fidelity In Vitro Packaging of Diverse Synthetic Cargo into Encapsulin Protein Cages

Abstract Cargo‐filled protein cages are powerful tools in biotechnology with demonstrated potential as catalytic nanoreactors and vehicles for targeted drug delivery. While endogenous biomolecules can be packaged into protein cages during their expression and self‐assembly inside cells, synthetic cargo molecules are typically incompatible with live cells and must be packaged in vitro. Here, we report a fusion‐based in vitro assembly method for packaging diverse synthetic cargo into encapsulin protein cages that outperforms standard in cellulo assembly, producing cages with superior uniformity and thermal stability. Fluorescent dyes, proteins and cytotoxic drug molecules can all be selectively packaged with high efficiency via a peptide‐mediated targeting process. The exceptional fidelity and broad compatibility of our in vitro assembly platform enables generalisable access to cargo‐filled protein cages that host novel synthetic functionality for diverse biotechnological applications.