Rebois R Victor, Robitaille Mélanie, Pétrin Darlaine, Zylbergold Peter, Trieu Phan, Hébert Terence E
National Institute of Deafness and Other Communication Disorders and National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892, USA.
Methods. 2008 Jul;45(3):214-8. doi: 10.1016/j.ymeth.2008.06.006. Epub 2008 Jun 27.
A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100A of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an example of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex.
通过对绿色荧光蛋白进行突变，已创造出多种具有不同光谱特性的荧光蛋白。当这些蛋白被一分为二时，任何一个片段本身都不具有荧光，而且通过共表达互补的N端和C端片段也无法重新构建出荧光蛋白。然而，当这些片段与在细胞内相互结合的蛋白进行基因融合时，荧光蛋白的N端和C端片段就会聚集在一起，从而重新构建出荧光蛋白。可以用各种荧光素酶同工型的两个互补片段进行类似的蛋白质互补分析（PCA）。这使得这些分析成为检测活细胞中两种蛋白质相互作用的有用工具。当来自发光或荧光供体蛋白的能量分别非辐射地转移到荧光受体蛋白时，就会发生生物发光共振能量转移（BRET）或荧光共振能量转移（FRET）。只有当蛋白质彼此距离在100埃以内时，这种能量转移才会发生。因此，BRET和FRET也是检测活细胞中两种蛋白质相互作用的有用工具。通过将不同的蛋白质片段互补分析（PCA）与BRET或FRET相结合，有可能证明在活细胞中三种或更多种蛋白质同时是同一蛋白质复合物的组成部分。作为这种方法实用性的一个例子，我们展示了多达四种不同的蛋白质作为G蛋白偶联受体信号复合物的一部分同时相互作用。
