Rapid and High-Sensitivity Cell-Based Assays of Protein-Protein Interactions Using Split Click Beetle Luciferase Complementation: An Approach to the Study of G-Protein-Coupled Receptors
Rapid and High-Sensitivity Cell-Based Assays of Protein-Protein Interactions Using Split Click Beetle Luciferase Complementation: An Approach to the Study of G-Protein-Coupled Receptors
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DOI：
10.1021/ac100104q
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发表时间：
2010-03-15
影响因子：
7.4
通讯作者：
Ozawa, Takeaki
中科院分区：
文献类型：
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作者：
Misawa, Naomi;Kafi, A. K. M.;Ozawa, Takeaki
为了在基础生物学和药物发现过程中识别生物相关化合物，用于阐明蛋白质-蛋白质相互作用的快速定量方法已变得必要。我们描述了一种新的光学技术，用于监测活细胞中的蛋白质-蛋白质相互作用的基础上，从点击甲虫（巴西Pyrearinus termitilluminans）的分裂荧光素酶片段的互补。利用半理性文库筛选鉴定了一对新的氨基端和羧基端荧光素酶片段，证明获得了显著更高的灵敏度和信号背景比。将鉴定的片段应用于与质膜上的β-抑制蛋白相互作用的五种G蛋白偶联受体（GPCR）的研究。通过产生稳定表达GPCR和与荧光素酶片段连接的β-抑制蛋白的细胞系，我们证明了使用96孔微量滴定板格式快速和灵敏地筛选作用于GPCR的潜在化学物质。筛选时间减少到5-10分钟后，配体刺激。最大响应变得比背景信号高15倍以上。这种荧光素酶互补方法也使得能够使用生物发光显微镜对单个活细胞中的相互作用进行准确的空间和时间分析。这些GPCR分析将促进多孔板形式的高通量筛选系统的开发。此外，使用感兴趣的特定蛋白质，荧光素酶的新片段将为活细胞和动物中许多细胞内信号的研究提供不同的测定方法。
To identify biologically relevant compounds in basic biology and drug discovery processes, rapid quantitative methods for elucidating protein-protein interactions have become necessary. We describe a novel optical technique for monitoring protein-protein interactions in living cells based on complementation of split luciferase fragments from click beetle (Brazilian Pyrearinus termitilluminans). A new pair of amino-terminal and carboxy-terminal fragments of the luciferase was identified using semirational library screening, demonstrating achieved markedly higher sensitivity and signal-to-background ratio. The identified fragments were applied to the study of five G-protein coupled receptors (GPCR) that interact with beta-arrestin on the plasma membrane. By generating cell lines stably expressing the GPCRs and beta-arrestin connected with the luciferase fragments, we demonstrated rapid and sensitive screening of potential chemicals that act on GPCRs using a 96-well microtiter plate format. The screening time was reduced to 5-10 min after ligand stimulation. The maximum response became more than 15-fold higher than the background signal. This luciferase complementation method also enabled accurate spatial and temporal analyses of interactions in single living cells using bioluminescence microscopy. These GPCR assays will facilitate developments of high-throughput screening systems in a multiwell plate format. Furthermore, using specific proteins of interest, the novel fragments of luciferase will provide different assay methods for the study of many intracellular signals in living cells and animals.