# Recent Advances in Protein Caging Tools for Protein Photoactivation
## 1 Introduction  
Methods for photo-regulating the function and quantity of proteins are attracting attention in a wide range of fields, from fundamental research in molecular biology [1; 2; 3; 4; 5] to drug delivery systems [6; 7; 8; 9]. In these methods, proteins are activated [10; 11; 2; 3; 4; 5; 2; 12] or released from their carriers in response to light irradiation in and around cells [6; 7; 8; 9; 13; 14]. Due to the high spatial and temporal resolution of light, the function of the protein can be selectively exerted at the desired location and at the desired time. The amount of active protein expressed can be remotely controlled by light. Therefore, we can obtain spatiotemporal information on the function of proteins by controlling the location and timing of light irradiation, giving insight into the dynamic roles of proteins in biological systems. Furthermore, light-induced activation and release of proteins are expected to reduce the off-target effects of protein-based drugs, and moreover potentially enable therapeutic effects to be obtained by producing only the minimum amount of therapeutic proteins selectively in the affected area.  
In recent years, optogenetics based on naturally occurring light-responsive proteins has been widely reported as a method for photo-regulation of proteins [15; 16; 17; 3; 4; 18]. Light-dependent interactions and structural changes of light-sensing proteins such as cryptochrome-2 [16], vivid [17] and phototropin [18], which are derived from plants and fungi, have been used to control the activity of functional proteins, including membrane receptors [16], gene-editing enzymes [17] and antibodies [18]. In these methods, a light-responsive protein that is genetically engineered is expressed as a fusion protein consisting of a functional protein of interest and a light-sensing domain such as the LOV domain of phototropin, which is the most familiar in optogenetics [18]. Since all parts of the protein can be expressed genetically, it is simple to express the desired photo-responsive proteins in living cells by the introduction of the gene using plasmid DNA or a virus vector. Inthe case that the protein of interest is activated by dimerization or complexation, it is relatively easy to design the photoactivatable fusion proteins. However, for many other proteins, the proteins of interest must be engineered elaborately to introduce optogenetics through reversibly partitioning them, in which the partitioned proteins are inactive before light-induced dimerization or complexation [17]. In addition, after photoactivation, the optogenetically activated proteins are kept to fuse to light-responsive domains, and such fusion with exogenous ones may reduce the activity and limit the range of applications. Recently, a photocleavable protein has been reported as a new category of optogenetics [19; 20]. In the pioneering report, the protein of interest was tethered to the membrane through the photocleavable protein, and after exposure to light, the active protein without any fusion partner was released from the membrane, leading to light-induced localization and activation [19]. This approach can simply design light-induced release of wild-type protein from specific organelle by fusion with organelle-localizing proteins through the photo-cleavable protein, but there are also many proteins whose activity cannot be suppressed before exposure to light by control of localization alone. Thus, although optogenetics is an extremely attractive tool for intracellular protein photoactivation, it may not be suitable for some applications in terms of convenience and versatility. The present review mainly focuses on chemical modification-based methodologies for protein photoactivation. As a complementary approach to optogenetics, the progress in the synthetic and semi-synthetic approaches for protein photoactivation are systematically introduced to help the researchers in a variety of fields to simply design and realize a suitable protein photoactivation system for their goals.