| Budget Amount *help |
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2012: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2011: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
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| Research Abstract |
In vitro synthesis of G protein coupled receptors (GPCRs) was examined to develop experimental systems useful for single-molecule fluorescence measurements. Firstly, lipid nanodiscs proteins were prepared as a platform for folding of GPCRs. Secondary, β2 adrenergic receptors were synthesized in insect-derived cell-free translational system in the presence of liposomes and ligand-binding ability was assessed by binding of fluorescent ligand. However, a commercially available fluorescent ligand had a significant nonspecific binding to lipid membranes. To find fluorescent ligands with little nonspecific binding, Cy3- and Alexa647- labeled noradrenaline were synthesized. Both Cy3-NA and Alexa647-NA did not have sufficient binding affinity to the receptors that were expressed on CHO cells, suggesting importance of further development of probes.
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