# Recent Advances in Protein Caging Tools for Protein Photoactivation
## 3 Site-Specific Protein Caging
phosphotyrosine [46] have been utilized for photo-activation of various proteins including enzymes [35; 39; 42; 43; 44; 45], intein proteins [36; 47], ion channels [37; 38; 40], receptors [46], phosphorylation cascade proteins [50; 51], and so on [5; 48; 49; 52; 53]. The site-specific incorporation of unnatural caged amino acid into proteins was first achieved in _E. coli_[35; 36; 42; 43; 44] and a cell-free protein expression system [46], and subsequently their photoactivation was examined in vitro. Furthermore, the recombinant caged proteins were transduced into mammalian cells with a transfection reagent to utilize their photoactivatable functions for clarifying the spatial and temporal molecular mechanism of the living system [5; 48]. Next, to photo-regulate ion channels on the plasma membrane of living cells, in situ expression of site-specifically caged proteins was performed in _Xenopus_ oocytes by injecting the caged amino acids-combined tRNA through microinjection [37; 38; 40]. Recently, the genetic code extension technology has become applicable to eukaryotic cells by using the genetic expression system for the orthogonal pair of pyrrolysyl-tRNA synthetase and the corresponding tRNA, enabling the genetic incorporation of caged amino acids into proteins in yeast cells [42; 51] and mammalian cells [5; 49; 50; 51; 52; 53]. By using such a genetic encoding system, a pioneer group, [PERSON] et al. demonstrated light-induced intracellular localization change of caged nuclear localization proteins in human cells [49] and achieved photoactivation of receptor-mediated signal transduction by caging a phosphorylation protein, STAT1 [50]. Furthermore, to understand the role of cancer-specific mutation, they reported to photoactivate the synthesis of the onometabolite (\(R\))-2-hydroxyglutamate through caging an isocitrate dehydrogenase mutant in normal cells and exposing those engineered cells to light [52]. Such genetic caging with unnatural amino acid introduction was also reported to achieve light-induced gene editing through caging of Cas9 [53], photo-activation of immune response through caging of MEK1 [5], and screening of caspase substrate proteins through caging of caspase-3 [5] in human cells. Thus, in this approach, a variety of wild-type proteins could be site-specifically caged by utilizing various caged amino acids. Moreover, this approach is advantageous in introducing a photodegradable protection into the active site inside the closed structure of proteins because the caged amino acid can be incorporated on translation before protein folding. However, it requires advanced and specialized gene engineering techniques, and therefore is not easily available. Other strategies in protein caging have also been reported. In semisynthetic approaches, a small part of the protein was chemically synthesized by incorporating caged amino acid through peptide synthesis, and then linked with a major part of the protein to produce a site-specifically caged protein [54; 55; 56]. First, as a pioneering approach, site-specific caged ribonuclease S (RNase S) was prepared by mixing a synthesized caged S-peptide and S-protein [54]. RNase S is consisting of two peptide fragments, S-peptide (1-20 residues) and S-protein (21-124 residues), and these fragments bind in a self-assembly manner to exert the enzymatic activity. In this report, by optimizing the replacement site of caged amino acids in S-peptide, the caged RNase S was activated by light in an off-on manner [54]. This approach was greatly developed by the expressed protein ligation (EPL) method (Figure 2C) [55; 56]. In EPL-based caging, the fusion protein of a C-terminally-truncated target protein with a self-processing intetin domain is overexpressed in _E. coli_, and through the intein-mediated processing reaction [57], the C-terminal is converted to a reactive thioester moiety. In parallel, the C-terminal domain peptide including a cysteine residue at the \(N\)-terminal end and caged amino acids at the desired positions is chemically synthesized.