Intein-Mediated Reconstitution of Split Lumazine Synthase for Programmable Protein Nanocage Assembly

Background/Objectives: Protein nanocages are versatile platforms with potential applications in drug delivery, enzyme encapsulation, and bioreactor systems, owing to their precise self-assembly and excellent biocompatibility. However, most protein cage systems have limited accessibility to their internal space, which hinders the efficient encapsulation of large molecules or complex proteins. Methods and results: In this study, we propose a programmable reassembly system by artificially splitting the monomer of lumazine synthase, a protein that naturally forms a nanocage through self-assembly. Using intein-mediated protein splicing, the self-assembly of the monomer was converted into a condition-dependent reaction, enabling the incorporation of large or functional biomolecules prior to the assembly stage. Furthermore, to achieve targeted delivery, an EGFR-binding affibody (EGFRAfb) was fused to the split monomer so that it is exposed on the cage surface after reassembly, thereby providing selective binding capability toward EGFR-expressing cells. Successfully reassembled nanocages were visualized, and the fluorescent proteins encapsulated within them were delivered to the target and activated in specific cells. Conclusions: Therefore, the programmable protein nanoplatform presented in this study can overcome the spatial limitations of conventional protein cages while allowing for precise control over both the timing of cage assembly and targeted molecular delivery.