Kim Sung-Bae, Tao Hiroaki
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, 305-8569, Japan.
AIST Shikoku center, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa, 761-0395, Japan.
Methods Mol Biol. 2016;1461:165-73. doi: 10.1007/978-1-4939-3813-1_13.
The present protocol demonstrates a new strategy for imaging ligand-triggered protein phosphorylation using circularly permutated luciferases (cpLuc): (1) a luciferase is first fragmented into two segments for creating new N- and C-terminal ends in the hydrophilic region, (2) the original N- and C-terminal ends are circularly permutated and linked via a GS linker, whereas the new ends made by fragmentation are correspondingly linked with two proteins of interest. When the new ends of the cpLuc are linked with the ligand-binding domain of estrogen receptor (ER LBD) and Src homology two domain of Src (SH2), the estrogen can trigger phosphorylation of the ER LBD and consequent intramolecular ER LBD-SH2 binding. This interaction triggers an approximation of the adjacent fragments of split-cpLuc recovering the enzyme activity. This probe design greatly improves signal-to-noise (S/N) ratios upon tracing weak protein-protein interactions (PPIs) in mammalian cells.
本方案展示了一种使用环形排列荧光素酶（cpLuc）对配体触发的蛋白质磷酸化进行成像的新策略：（1）首先将荧光素酶片段化为两个片段，以便在亲水区创建新的N端和C端；（2）将原始的N端和C端进行环形排列，并通过GS接头连接，而通过片段化产生的新末端则相应地与两个感兴趣的蛋白质相连。当cpLuc的新末端与雌激素受体的配体结合域（ER LBD）和Src的Src同源2结构域（SH2）相连时，雌激素可触发ER LBD的磷酸化以及随后的分子内ER LBD-SH2结合。这种相互作用触发了分裂cpLuc相邻片段的靠近，从而恢复酶活性。这种探针设计在追踪哺乳动物细胞中微弱的蛋白质-蛋白质相互作用（PPI）时极大地提高了信噪比（S/N）。
