# Recent Advances in Protein Caging Tools for Protein Photoactivation
## 5 Photolytic Protein Aggregates  
As another strategy to make proteins photo-responsive, a method for the preparation of photolytic protein aggregates is introduced. Protein crystals and aggregates have been reported to be applied to solid-state enzymes and protein stabilization for thirty years [70,71],  
Figure 4: Sterically bulky caging of transferrin (Tf). (**A**) Fluorescence microscopy images of colon cancer DLD1 cells treated with intact Tf or sterically bulky caged Tf after exposure to light at 0 and 10 J/cm\({}^{2}\)[68]. (**B**) Schematic illustration of light-induced intracellular delivery of cargos by using sterically bulky caged Tf [68]. because they are highly stable against proteolysis and denaturation, and can accumulate an active protein inside them at a high concentration. Utilizing these advantages, protein aggregates, which are reversibly dissolved by stimuli, have been studied to be applied to protein delivery [72]. However, photolytic protein aggregates have not been reported. In the development of the sterically bulky caging method described above, we accidentally noticed that photolytic protein aggregates were generated when the number of SA equivalents to the biotinylated protein was reduced. We hypothesized that the aggregate formation was due to the polymerization of the multiply biotinylated protein through cross-linking with SA, which has four biotin-binding sites. Furthermore, we thought that it would be interesting to use the photolytic protein aggregates as a new tool for light-induced release of active proteins (Figure 5A) [73]. First, the procedures for the preparation of the aggregates were optimized by using biotinylated protected HEL and SA. When high concentrations of both components were mixed in a 1:1 ratio in aqueous solution, the aqueous solution started to become cloudy. By adding a large excess of free biotin molecules to stop the growth of aggregates via blocking the biotin-binding sites of SA, micrometer-sized aggregates were obtained (Figure 5B). No residual proteins were detected in the supernatant after centrifugation, indicating that almost all of HEL and SA were incorporated into the aggregates. When the aggregates were exposed to light, they dissolved according to the amount of exposed light (Figure 5C), and SA and HEL were released into the aqueous solution, as shown by electrophoretic analysis [73]. At maximum, 90% of the HEL used for aggregate formation was released without any modification. The bacteriolytic activity was also recovered up to 90% in the wild-type HEL used for aggregate formation. Thus, the specific activity of the photo-dissolved HEL from aggregates was the same as that of the wild type, suggesting that aggregate formation and photo-irradiation do not cause irreversible denaturation. Such photolytic aggregate formation and light-dependent dissolution were also observed with biotinylated protected Tf [73]. Similar to the sterically bulky caging described above, we were also able to form aggregates using Dox-modified Tf and confirm the induction of cell death in response to light exposure (Figure 5D). Thus, this method is also considered to be highly versatile. Next, we examined the processing method of the photolabile protein aggregates. When we simply mixed biotinylated protected proteins with SA as described above, micro-sized aggregates with non-uniform shape and size were produced. Furthermore, this size is too large to be intravenously administered in vivo, nor can it enter cells. To down-size the aggregates to the submicron rang, we mixed biotinylated HEL and SA in water/oil (\(w/o\)) emulsion droplets with submicron sizes. As a result, it was confirmed by electron microscopy that aggregates with a relatively uniform shape of several hundred nanometers in diameter could be prepared (Figure 5E) [73]. When such protein aggregates are utilized in living cells, they must have cell permeability.