Development of a technique for selective chemical labeling and modification of intracellular proteins

配体定向tosyl （LDT）化学是一种允许在细胞和体内对特定（天然）蛋白质进行选择性化学标记的技术。然而，LDT化学仍处于起步阶段，因此其潜力尚未得到充分开发。在这项工作中，我们首次尝试将LDT化学作为内源性蛋白质活细胞荧光成像的新工具。使用一种新设计的LDT试剂，我们能够在活的哺乳动物细胞中选择性地用有机荧光染料俄勒冈绿（OG）标记内源性FKBP12。通过FRET成像还可以看到细胞内og标记的FKBP12和dsred融合的FRB的雷帕霉素介导的络合。此外，在这项工作中，我们证明了将LDT化学与定点诱变技术相结合可以显著提高化学标记率和效率。

Ligand-directed tosyl (LDT) chemistry is a technique that allows selective chemical labeling of specific (native) proteins in cells and in vivo. However, the LDT chemistry is still at the beginning stage, and thus its full potential has yet to be exploited. In this work, we first attempted to apply the LDT chemistry as a novel tool for live-cell fluorescence imaging of endogenous proteins. Using a newly-designed LDT reagent, we were able to selectively label endogenous FKBP12 with an organic fluorescent dye, Oregon Green (OG), in living mammalian cells. It was also possible to visualize the rapamycin-mediated complexation of the OG-labeled FKBP12 and DsRed-fused FRB inside of the cells by FRET imaging. Furthermore, in this work, we demonstrated that the chemical labeling rate and efficiency could be significantly improved by combining the LDT chemistry with the site-directed mutagenesis technique.