Novel genetically encoded biosensors using firefly luciferase

被引:166
作者
Fan, Frank [1 ]
Binkowski, Brock F. [1 ]
Butler, Braeden L. [1 ]
Stecha, Peter F. [1 ]
Lewils, Martin K. [1 ]
Wood, Keith V. [1 ]
机构
[1] Promega Corp, Madison, WI 53711 USA
关键词
D O I
10.1021/cb8000414
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:346 / 351
页数:6
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