The present protocol introduces multicolor imaging of bifacial activities of an estrogen. For the multicolor imaging, the authors fabricated two single-chain probes emitting green or red bioluminescence (named Simer-G and -R, respectively) from click beetle luciferase (CBLuc) green and red: Simer-R consists of the ligand binding domain of estrogen receptor (ER LBD) and the Src homology-2 (SH2) domain of Src, which are sandwiched between split-CBLuc red (CBLuc-R). On the other hand, Simer-G emitting red light consists of the ER LBD and a common consensus sequence of coactivators (LXXLL motif), which are inserted between split-CBLuc green (CBLuc-G). This probe set creates fingerprinting spectra from the characteristic green and red bioluminescence in response to agonistic and antagonistic activities of a ligand of interest. The present protocol further provides a unique methodology to calculate characteristic estrogenicity scores of various ligands from the spectra.
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This work was supported by grants from Japan Society for the Promotion of Science (JSPS), grant numbers 26288088, 15KK0029, and 16K14051.
Kim SB, Awais M, Sato M, Umezawa Y, Tao H (2007) Integrated molecule-format bioluminescent probe for visualizing androgenicity of ligands based on the intramolecular association of androgen receptor with its recognition peptide. Anal Chem 79:1874–1880CrossRefPubMedGoogle Scholar
Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132CrossRefPubMedGoogle Scholar
Kim SB, Umezawa Y, Kanno KA, Tao H (2008) An integrated-molecule-format multicolor probe for monitoring multiple activities of a bioactive small molecule. ACS Chem Biol 3:359–372Google Scholar
Viviani VR, Uchida A, Viviani W, Ohmiya Y (2002) The influence of Ala243 (Gly247), Arg215 and Thr226 (Asn230) on the bioluminescence spectra and pH-sensitivity of railroad worm, click beetle and firefly luciferases. Photochem Photobiol 76:538–544CrossRefPubMedGoogle Scholar
Kim SB, Otani Y, Umezawa Y, Tao H (2007) Bioluminescent indicator for determining protein-protein interactions using intramolecular complementation of split click beetle luciferase. Anal Chem 79:4820–4826CrossRefPubMedGoogle Scholar
Kim SB, Ozawa T, Watanabe S, Umezawa Y (2004) High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase. Proc Natl Acad Sci U S A 101:11542–11547CrossRefPubMedPubMedCentralGoogle Scholar
Ozawa T, Takeuchi TM, Kaihara A, Sato M, Umezawa Y (2001) Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time. Anal Chem 73:5866–5874CrossRefPubMedGoogle Scholar