A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins
A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins
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DOI：
10.1073/pnas.96.1.151
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发表时间：
1999-01-05
影响因子：
11.1
通讯作者：
Johnson, CH
中科院分区：
文献类型：
--
作者：
Xu, Y;Piston, DW;Johnson, CH
我们描述了一种分析蛋白质相互作用的方法，它比以前的分析方法有一些吸引人的优点。这种方法被称为生物发光共振能量转移(BRET)，使用的是在基因上与一种候选蛋白质融合的生物发光荧光素酶，以及与另一种感兴趣的蛋白质融合的绿色荧光蛋白突变体。两种融合蛋白之间的相互作用可以使荧光素酶和绿色荧光蛋白足够近，从而发生共振能量转移，从而改变生物发光的颜色。通过利用蓝藻生物钟基因编码的蛋白质，我们使用Bret技术证明了时钟蛋白Kaib相互作用形成同源二聚体。BRET对于测试天然细胞内的蛋白质相互作用特别有用，特别是与完整的膜蛋白或针对特定细胞器的蛋白质的相互作用。
We describe a method for assaying protein interactions that offers some attractive advantages over previous assays. This method, called bioluminescence resonance energy transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and a green fluorescent protein mutant fused to another protein of interest. Interactions between the two fusion proteins can bring the luciferase and green fluorescent protein close enough for resonance energy transfer to occur, thus changing the color of the bioluminescent emission. By using proteins encoded hy circadian (daily) clock genes from cyanobacteria, we use the BRET technique to demonstrate that the clock protein KaiB interacts to form homodimers. BRET should be particularly useful for testing protein interactions within native cells, especially with integral membrane proteins or proteins targeted to specific organelles.