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
Naomi Misawa
A. K. M. Kafi
Mitsuru Hattori
Kenji Miura
Kenji Masuda
Takeaki Ozawa
10.1021/ac100104q.s002
https://acs.figshare.com/articles/media/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/2784355
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 <i>Pyrearinus termitilluminans</i>). 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 β-arrestin on the plasma membrane. By generating cell lines stably expressing the GPCRs and β-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.
2010-03-15 00:00:00
split luciferase fragments
microtiter plate format
cell lines stably
background signal
novel fragments
Click Beetle Luciferase Complementation
multiwell plate format
ligand stimulation
intracellular signals
GPCR assays
interaction
screening time
luciferase complementation method
drug discovery processes
semirational library screening
plasma membrane
assay methods
arrestin
click beetle
Brazilian Pyrearinus termitilluminans
luciferase fragments
protein
bioluminescence microscopy