Fluorogenic affinity label for the facile, rapid imaging of proteins in live cells
Fluorogenic affinity label for the facile, rapid imaging of proteins in live cells
复制标题
DOI：
10.1039/b907664f
复制
发表时间：
2009-01-01
影响因子：
3.2
通讯作者：
Raines, Ronald T.
中科院分区：
文献类型：
--
作者：
Watkins, Rex W.;Lavis, Luke D.;Raines, Ronald T.
卤代烷烃脱卤酶（HD）通过共价酶-底物中间体催化卤代烷烃的水解。将靶蛋白与不能水解中间体的HD变体融合使得能够在纤维素中用卤代烷标记靶蛋白。然而，现有探针的实用性受到背景荧光以及有限的膜渗透性的阻碍。在这里，我们报告的合成和使用的荧光亲和标记，通过细胞内酯酶揭露后，标签的HD变异体在cellulo。标记是快速和具体的，从依赖酶催化剂和高膜渗透性的探针之前和之后解蔽预期。最值得注意的是，即使是高浓度的荧光亲和标记物也会导致最小的背景荧光，而无需洗涤细胞。我们设想，这种荧光亲和标记，其中争取催化两种细胞酶，将发现实用程序中的脉冲追踪实验，高内容筛选，和许多其他协议。
Haloalkane dehalogenase (HD) catalyzes the hydrolysis of haloalkanes via a covalent enzyme-substrate intermediate. Fusing a target protein to an HD variant that cannot hydrolyze the intermediate enables labeling of the target protein with a haloalkane in cellulo. The utility of extant probes is hampered, however, by background fluorescence as well as limited membrane permeability. Here, we report on the synthesis and use of a fluorogenic affinity label that, after unmasking by an intracellular esterase, labels an HD variant in cellulo. Labeling is rapid and specific, as expected from the reliance upon enzymic catalysts and the high membrane permeance of the probe both before and after unmasking. Most notably, even high concentrations of the fluorogenic affinity label cause minimal background fluorescence without a need to wash the cells. We envision that such fluorogenic affinity labels, which enlist catalysis by two cellular enzymes, will find utility in pulse-chase experiments, high-content screening, and numerous other protocols.