Novel genetically encoded biosensors using firefly luciferase
Novel genetically encoded biosensors using firefly luciferase
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DOI：
10.1021/cb8000414
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发表时间：
2008-06-01
影响因子：
4
通讯作者：
Wood, Keith V.
中科院分区：
文献类型：
--
作者：
Fan, Frank;Binkowski, Brock F.;Wood, Keith V.
基因编码生物传感器已被证明对于实时监测细胞内现象很有价值，特别是结合了绿色荧光蛋白变体的基于 FRET 的传感器。为了提高检测灵敏度和响应动态，我们对萤火虫荧光素酶进行了基因改造，通过调节其发光活性来检测特定的分子间相互作用。这一概念已应用于共价、非共价和变构设计配置。共价设计通过切割依赖性的发光增加来灵敏地检测蛋白酶活性。非共价和变构设计可分别实现小分子雷帕霉素和 cAMP 的可逆检测。这些传感器允许在将荧光素底物添加到生长培养基中后检测活细胞内的分子过程。例如，CAMP 传感器可以监测与 G 蛋白偶联受体功能相关的细胞内信号转导。这些和其他发光生物传感器将可用于研究和药物发现中细胞生理学的灵敏检测。
Genetically encoded biosensors have proven valuable for real-time monitoring of intracellular phenomena, particularly FRET-based sensors incorporating variants of green fluorescent protein. To increase detection sensitivity and response dynamics, we genetically engineered firefly luciferase to detect specific intermolecular interactions through modulation of its luminescence activity. This concept has been applied in covalent, noncovalent, and allosteric design configurations. The covalent design gives sensitive detection of protease activity through a cleavage-dependent increase in luminescence. The noncovalent and allosteric designs allow reversible detection of the small molecules rapamycin and cAMP, respectively. These sensors allow detection of molecular processes within living cells following addition of the luciferin substrate to the growth medium. For example, the CAMP sensor allows monitoring of intracellular signal transduction associated with G-protein coupled receptor function. These and other luminescent biosensors will be useful for the sensitive detection of cellular physiology in research and drug discovery.