Incorporation of thrombin cleavage peptide into a protein cage for constructing a protease-responsive multifunctional delivery nanoplatform.

Protein cages are spherical hollow supramolecules that are attractive nanoscale platforms for constructing cargo delivery vehicles. Using ferritin isolated from the hyperthermophilic archaeon Pyrococcus furiosus (Pf_Fn), we developed a multifunctional protein cage-based delivery nanoplatform that can hold cargo molecules securely, deliver them to the targeted sites, and release them to the targeted cells. The release is triggered by cleavage induced by the protease, thrombin. The thrombin cleavage peptide (GGLVPR/GSGAS) was inserted into the flexible loop region of Pf_Fn, which is located at a 4-fold axis of symmetry exposed on the surface of protein cages (Thr-Pf_Fn). Subsequently, the C-terminal glycine, which is situated in the interior cavity, was substituted with cysteine (G173C) to permit site-specific conjugation of cargo molecules. The introduced cysteine (G173C) was labeled with a fluorescent probe (F5M-Thr-Pf_Fn) for cell imaging and cargo release monitoring. The surface of F5M-Thr-Pf_Fn was fur...