# Recent Advances in Protein Caging Tools for Protein Photoactivation
## 4 Sterically Bulky Caging of Proteins  
In order to solve the problems of caged proteins, we developed a sterically bulky caging method [66]. Among conventional protein caging methods, the approach of randomly modifying amino groups on the protein surface has great advantages in terms of simplicity and versatility, as long as the inhibitory effect on the function can be improved. In order to improve the suppression of any protein functions before photodegradation, we aimed to increase the steric hinderance of each protecting group. If most of the protein surface can be hidden with the randomly modified protecting groups, the interaction at the functional site of the protein will surely be suppressed. This was our strategy at the beginning of this study. At that time, in another project, we had been studying the introduction of photodegradable protecting groups neighboring to promoter regions as a site-specific caging technique for DNA plasmids. In that project, we had developed a simple method of bulking up the protecting group to protect the adjacent promoter [67]. A biotin-modified photodegradable protecting group was developed and introduced neighboring to the promoter, and then, the caged plasmid was mixed with streptavidin (SA), a bulky biotin-binding protein consisting of tetramers. In this study, the bulky SA molecule was confirmed to attach on the introduced biotin moiety on the plasmid and efficiently suppress transcription, probably due to the steric hinderance against the binding of transcription factors onto the neighboring promoter region [67]. Therefore, we hypothesized that this sterically bulky caging approach using biotinylated caging reagent (BCR) and SA could be applied to random caging of proteins (Figure 3A). First, we used hen egg lysozyme (HEL), a bacterolytic enzyme, as a model protein and randomly introduced biotinylated photodegradable protecting groups with BCR [66]. Mass spectrometry analysis showed that one to three protecting groups were introduced per HEL molecule. When the biotinylated protected HEL was mixed with SA (more than eight equivalents), electrophoretic analysis confirmed the preparation of a bulky caged HEL consisting of one HEL and one to three SA bound. The bacteriolytic activity of the caged HELs was found to be 80% of that of the untreated HELs when only the small biotinylated protective groups were modified. On the other hand, after SA binding, the activity decreased to 3.6% (Figure 3B) [66]. This result indicates that the sterically bulky caging approach using the steric hindrance of SA is very effective in inhibiting the activity. When the bulky caged HELs were exposed to light, the protective groups were decomposed, and electrophoretic analysis confirmed that the unmodified HELs were generated according to the amount of exposed light. The activity after light exposure was recovered to more than 70% (Figure 3B). Thus, it was shown that photoactivatable proteins can be easily prepared by sterically bulky caging using BCR and SA [66]. To demonstrate the versatility of the sterically bulky caging method, we applied it to transferrin (Tf), a human iron transport protein [68]. When the bulky caged Tf was applied to cultured cells, its binding to the Tf receptor on the cell surface was inhibited and it was not taken up into the cells. After light irradiation, it was taken up into the cells in response to light, as confirmed by confocal laser microscopy (Figure 4A) [68]. Thus, this method was applicable to the light-induced control of a transporter protein on living cells without any significant damage onto the cells. Here, Tf has been employed as a carrier for intracellular drug delivery, and when Tf modified with a drug via a linker that cleaves intracellularly is applied to cells, this leads to accumulation of drugs in the cells [69]. Therefore, an apoptosis-inducing drug, doxorubicin (Dox), was installed onto Tf as a model cargo  
Figure 3: Sterically bulky caging of proteins.