Abstract
The present protocol introduces a single-chain probe carrying a functional peptide in the N-terminal domain of the androgen receptor (AR NTD) for illuminating androgenicity of ligands. In the single-chain probe, a functional peptide in the AR NTD was genetically fused to the ligand-binding domain of AR (AR LBD) via a flexible linker, and then sandwiched between the N- and C-terminal fragments of split-firefly luciferase (FLuc) dissected at D415. This single-chain probe exerts (1) a high signal-to-background ratio and (2) sensitive discrimination between agonists and antagonists, where the dimerization of AR LBD is not involved. The present protocol guides a fundamental methodology on how to discriminate weak protein–protein (peptide) binding, and provides a new insight into the intramolecular folding inside monomeric AR.
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Acknowledgements
This work was supported by grants from Japan Society for the Promotion of Science (JSPS), grant numbers 26288088, 16K14051, and 15KK0029.
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Kim, SB., Tao, H. (2016). Single-Chain Probes for Illuminating Androgenicity of Chemicals. In: Kim, S. (eds) Bioluminescence. Methods in Molecular Biology, vol 1461. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3813-1_11
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DOI: https://doi.org/10.1007/978-1-4939-3813-1_11
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-3813-1
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