title: Single-Chain Probes for Illuminating Androgenicity of Chemicals.
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.
authors.affiliation: Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, 305-8569, Japan. kimu-sb@aist.go.jp.
authors.forename: Sung-Bae
authors.initials: SB
authors.lastname: Kim
authors.affiliation: AIST Shikoku center, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa, 761-0395, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tao
mesh_terms: D000595:Amino Acid Sequence; D000728:Androgens / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D003412:Cricetulus; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008163:Luminescent Measurements / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods; D011485:Protein Binding; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D011944:Receptors, Androgen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000728:Androgens; D008024:Ligands; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins
keywords: Androgen receptor; Androgenicity; Bioluminescence; Firefly luciferase; Single-chain probe