Engineering protein nanocages as carriers for biomedical applications

Bhaskar, Sathyamoorthy; Lim, Sierin

The interface between subunits of viruses such as CCMV was studied to determine the mechanism of supramolecular assembly of protein nanocages. 77 In-depth study of the interactions between protein subunits at the interface has paved the way to optimizing the conditions for the induction of self-assembly. Genetic modification of specific locations on the subunits, especially at the N and C termini, yields VLPs with different sizes and total number of subunits. 77 The CCMV viral cage was also tested in terms of low symmetrical assembly. Two different strategies were developed to break the symmetry of ligand presentation on the virus. 5 CCMV nanoparticles were labeled differently with either biotin or digoxigenin and then were disassembled into individual subunits ( Figure 13 ). These differentially labeled subunits were purified, mixed and reassembled in optimal stoichiometry to form dual-function nanocages with controlled display of ligands. The maximum benefit of this strategy can be reaped when a functional nanocage structure is assembled based on a library of differentially engineered subunits, each having its own ligand or epitope. 5 Protein-protein interactions often involve large areas of buried surface that can be interrupted by small molecules that target Engineered protein cages in biomedical applications S Bhaskar and S Lim 'hot spots' where the energy for binding is concentrated. 78 Zhang et al. 78 identified these hot spots in DNA-binding protein from starved cell by the alanine shaving method. This method works by conceptually shaving the individual side chains to methyl residues; the stabilities of the resulting mutants are deduced by comparing them with the wild type. This work could aid the basic understanding of the assembly of miniferritin and the control of ferritin's oligomerization state, which can be applied as templates for nanomaterial synthesis and drug delivery. By modifying the interactions at the subunit-subunit interface, the assembly profile of E2 protein nanocages can be modified such that they are stable at pH 7.4 but disintegrate at acidic pH. 43, 79 In other words, the pH transition point at which this change occurs can be controlled. Using this strategy, the release of the encapsulated drug/active molecules can be achieved by modulating the interactions between the subunits to respond to pH changes in the target cell microenvironment. 79 For example, the tumor microenvironment is slightly more acidic than healthy tissues. pH changes are also experienced by molecules entering cells by endocytosis. The molecules will experience a pH level of 7 near the cells and then a pH level of 5 upon entering the lysosome later in the pathway. Thus, pH-based assembly and disassembly responds could be used to trigger the release of molecules encapsulated within protein nanocages.