One-Pot Orthogonal Dual Functionalization of mi3 Self-Assembling Protein Nanoparticles via Sortase A and SpyCatcher/SpyTag Ligation

We present a one-pot approach for orthogonal dual functionalization of mi3 self-assembling protein nanoparticles by combining SpyCatcher003/SpyTag003 isopeptide bond formation with Sortase A-mediated transpeptidation. The nanoparticles were engineered with N-terminal SpyCatcher003 domains and C-terminal LPETGGH motifs, enabling site-specific conjugation of distinct cargo molecules. To overcome expression limitations, an N-terminal vesicle nucleating peptide (VNP6) was incorporated, which enhanced protein yields while maintaining nanocage integrity. Critical to this strategy, the calcium-independent Sortase A variant (SRT7+) was employed to avoid calcium-induced nanoparticle aggregation, enabling orthogonal ligation under physiological conditions. The platform enabled simultaneous conjugation of HER2-targeting affibodies and fluorescent probes in a single reaction, and these dual-functionalized self-assembling protein nanoparticles exhibited specific cellular uptake in HER2-positive cancer cells. This integrated one-pot approach proved scalable and modular for engineering protein-based nanomaterials for targeted therapy and diagnostics.