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Single-Chain Probes for Illuminating Androgenicity of Chemicals

  • Sung-Bae KimEmail author
  • Hiroaki Tao
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1461)

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.

Key words

Single-chain probe Bioluminescence Androgen receptor Androgenicity Firefly luciferase 

Notes

Acknowledgements

This work was supported by grants from Japan Society for the Promotion of Science (JSPS), grant numbers 26288088, 16K14051, and 15KK0029.

References

  1. 1.
    Guermeur Y, Geourjon C, Gallinari P, Deleage G (1999) Improved performance in protein secondary structure prediction by inhomogeneous score combination. Bioinformatics 15:413–421CrossRefPubMedGoogle Scholar
  2. 2.
    Dubbink HJ, Hersmus R, Verma CS, van der Korput HA, Berrevoets CA, van Tol J, Ziel-van der Made AC, Brinkmann AO, Pike AC, Trapman J (2004) Distinct recognition modes of FXXLF and LXXLL motifs by the androgen receptor. Mol Endocrinol 18:2132–2150CrossRefPubMedGoogle Scholar
  3. 3.
    Kim SB, Umezawa Y, Tao H (2009) Determination of the androgenicity of ligands using a aingle-chain probe carrying androgen receptor N-terminal peptides. Anal Sci 25:1415–14204Google Scholar
  4. 4.
    Langley E, Zhou ZX, Wilson EM (1995) Evidence for an anti-parallel orientation of the ligand-activated human androgen receptor dimer. J Biol Chem 270: 29983–29990Google Scholar
  5. 5.
    He B, Kemppainen JA, Wilson EM (2000) FXXLF and WXXLF sequences mediate the NH2-terminal interaction with the ligand binding domain of the androgen receptor. J Biol Chem 275: 22986–22994Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Research Institute for Environmental Management TechnologyNational Institute of Advanced Industrial Science and Technology (AIST)TsukubaJapan
  2. 2.AIST Shikoku centerNational Institute of Advanced Industrial Science and Technology (AIST)TakamatsuJapan

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