期刊
ANALYTICAL CHEMISTRY
卷 82, 期 6, 页码 2552-2560出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac100104q
关键词
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资金
- Japan Society for the Promotion of Science (JSPS)
- Japan Science and Technology Corporation (JST)
- New Energy Industrial Technology Development Organization (NEDO)
- Global COE Program
- MEXT, Japan [S0801035]
- Kato Memorial Foundation
- Grants-in-Aid for Scientific Research [21675001] Funding Source: KAKEN
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.
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