Circular Permutation Probes for Illuminating Phosphorylation of Estrogen Receptor

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


The present protocol demonstrates a new strategy for imaging ligand-triggered protein phosphorylation using circularly permutated luciferases (cpLuc): (1) a luciferase is first fragmented into two segments for creating new N- and C-terminal ends in the hydrophilic region, (2) the original N- and C-terminal ends are circularly permutated and linked via a GS linker, whereas the new ends made by fragmentation are correspondingly linked with two proteins of interest. When the new ends of the cpLuc are linked with the ligand-binding domain of estrogen receptor (ER LBD) and Src homology two domain of Src (SH2), the estrogen can trigger phosphorylation of the ER LBD and consequent intramolecular ER LBD–SH2 binding. This interaction triggers an approximation of the adjacent fragments of split-cpLuc recovering the enzyme activity. This probe design greatly improves signal-to-noise (S/N) ratios upon tracing weak protein–protein interactions (PPIs) in mammalian cells.

Key words

Circular permutation Phosphorylation Luciferase Estrogen receptor Bioluminescence 



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


  1. 1.
    Weissleder R, Ntziachristos V (2003) Shedding light onto live molecular targets. Nat Med 9:123–128CrossRefPubMedGoogle Scholar
  2. 2.
    Kim SB (2012) Labor-effective manipulation of marine and beetle luciferases for bioassays. Protein Eng Des Sel 25:261–269CrossRefPubMedGoogle Scholar
  3. 3.
    Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132CrossRefPubMedGoogle Scholar
  4. 4.
    Kotlikoff MI (2007) Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology. J Physiol 578:55–67CrossRefPubMedGoogle Scholar
  5. 5.
    Souslova EA, Chudakov DM (2007) Genetically encoded intracellular sensors based on fluorescent proteins. Biochemistry (Mosc) 72:683–697CrossRefGoogle Scholar
  6. 6.
    Kim SB, Sato M, Tao H (2008) Circularly permutated bioluminescent probes for illuminating ligand-activated protein dynamics. Bioconjug Chem 19:2480–2486CrossRefPubMedGoogle 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)Takamatsu, KagawaJapan

Personalised recommendations