C‐terminal engineered human H‐chain ferritin for peptide loading within the protein nanocage

Self‐assembling proteins are widely used in bionanotechnology due to their biocompatibility, non‐toxicity, structural stability, and easy functionalization. Ferritins (Fts) are particularly attractive systems, as they assemble into highly ordered nanocages with a hollow internal cavity. Their assembly and disassembly can be modulated by various protocols, such as changes in pH or the use of denaturing agents, enabling their application in molecular encapsulation and drug delivery. However, the efficiency of encapsulation remains challenging, often resulting in low or heterogeneous loading and incomplete protein recovery. Here, we load peptides into the core of the human H‐chain ferritin ( hHFt ) by genetically fusing them to the C‐terminus of the Ft subunit, a region facing the inner cavity. As model peptides, we used fragments derived from the SARS ‐ CoV ‐2 Spike protein sharing similar physicochemical properties but different charges. The structure of the functionalized nanocages was assessed using techniques like transmission electron microscopy, native gel electrophoresis, and X‐ray crystallography. High‐resolution structural analysis revealed interesting conformational changes in the hHFt BC ‐loop associated with C‐terminal functionalization, while the overall nanocage architecture is preserved. Additionally, NMR measurements of the transverse relaxation ( T 2 ) of a target peptide demonstrated that the nanocages do not expose the fused peptides under native conditions, but upon disassembly, can efficiently expose them, demonstrating that C‐terminal genetic functionalization of ferritin nanocages is a robust and effective approach for nanobiotechnological applications.