Kim Sung-Bae, Tao Hiroaki
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, 305-8569, Japan.
AIST Shikoku center, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa, 761-0395, Japan.
Methods Mol Biol. 2016;1461:143-51. doi: 10.1007/978-1-4939-3813-1_11.
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.
