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Bioluminescent Imaging of MAPK Function with Intein-Mediated Reporter Gene Assay

  • Akira Kanno
  • Takeaki Ozawa
  • Yoshio Umezawa
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 574)

Abstract

For nondestructive analysis of chemical processes in living mammalian cells, here we show a new reporter gene assay for detecting Ras–Raf-1 interactions based on protein splicing of transcription factors with DnaE inteins. The protein splicing induces connection of a DNA-binding protein (modified LexA; mLexA) with a transcription activation domain of a herpes simplex virus protein (VP16AD). Ras is connected with N-terminal DnaE and mLexA, while Raf-1 is connected with C-terminal DnaE and VP16AD. Upon stimulation with EGF, the interaction between Ras and Raf-1 triggers folding of the DnaEs, thereby inducing protein splicing to form mLexA–VP16AD fusion protein and transcription of a reporter gene, firefly luciferase. The extent of Ras–Raf-1 interaction is quantified by measuring the luciferase activity. By using the protein-splicing elements and the reporter gene, the Ras–Raf-1 interaction close to cell membranes can be evaluated.

Key words

Luciferase intein reporter gene transcription factor 

Notes

Acknowledgments

This work was supported by grants from the Japan Science and Technology Agency (JST), and Japan Society for the Promotion of Science (JSPS).

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Akira Kanno
    • 1
  • Takeaki Ozawa
    • 1
  • Yoshio Umezawa
    • 1
  1. 1.Department of Chemistry, School of ScienceThe University of Tokyo; Japan Science and Technology AgencyTokyoJapan

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