BRET3: a red-shifted bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein-protein interactions from single live cells and living animals
BRET3: a red-shifted bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein-protein interactions from single live cells and living animals
复制标题
DOI：
10.1096/fj.08-118919
复制
发表时间：
2009-08-01
期刊：
影响因子：
4.8
通讯作者：
Gambhir, Sanjiv Sam
中科院分区：
文献类型：
--
作者：
De, Abhijit;Ray, Pritha;Gambhir, Sanjiv Sam
利用生物发光共振能量转移（BRET）现象，我们报告了一个高光子效率，自发光融合蛋白结合突变红色荧光蛋白（mOrange）和突变海肾荧光素酶（RLuc 8）的发展。与现有的BRET融合蛋白相比，这种新的BRET融合蛋白（BRET 3）在光强度方面表现出数倍的改善。BRET 3还表现出利用其天然底物腔肠素的任何报道的生物发光蛋白质的最红移的光输出（564 nm峰值波长），其益处在小动物的各种组织深度中得到证实。BRET 3在单细胞水平上的成像效用使用分子内传感器证明，该分子内传感器结合了仅在雷帕霉素存在下二聚化的两种哺乳动物雷帕霉素途径蛋白质（FKBP 12和FRB）的靶。凭借其增加的光子强度，红移光输出和良好的光谱分辨率（类似于85 nm），BRET 3显示出改善的空间和时间分辨率，用于测量单细胞和活的小动物模型中的细胞内事件。进一步开发基于BRET 3的测定将允许使用单一测定对蛋白质-蛋白质相互作用进行成像，可直接从完整的活细胞扩展到小型活体受试者，从而加速药物发现。De，A.，Ray，P.，Loening，A. M.，甘比尔S. - 你好基于红移生物发光共振能量转移（BRET）的集成平台，用于对来自单个活细胞和活动物的蛋白质-蛋白质相互作用进行成像。FASEB J. 23，2702-2709（2009）
Taking advantage of the bioluminescence resonance energy transfer (BRET) phenomenon, we report the development of a highly photon-efficient, self-illuminating fusion protein combining a mutant red fluorescent protein (mOrange) and a mutant Renilla reniformis luciferase (RLuc8). This new BRET fusion protein (BRET3) exhibits severalfold improvement in light intensity in comparison with existing BRET fusion proteins. BRET3 also exhibits the most red-shifted light output (564-nm peak wavelength) of any reported bioluminescent protein that utilizes its natural substrate coelenterazine, a benefit of which is demonstrated at various tissue depths in small animals. The imaging utility of BRET3 at the single-cell level is demonstrated using an intramolecular sensor incorporating two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. With its increased photon intensity, red-shifted light output, and good spectral resolution (similar to 85 nm), BRET3 shows improved spatial and temporal resolution for measuring intracellular events in single cells and in living small animal models. The development of further BRET3-based assays will allow imaging of protein-protein interactions using a single assay directly scalable from intact living cells to small living subjects, allowing accelerated drug discovery.-De, A., Ray, P., Loening, A. M., Gambhir, S. S. BRET3: a red-shifted bioluminescence resonance energy transfer ( BRET) based integrated platform for imaging protein-protein interactions from single live cells and living animals. FASEB J. 23, 2702-2709 ( 2009)