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A Protein–Protein Interaction Assay FlimPIA Based on the Functional Complementation of Mutant Firefly Luciferases

  • Yuki Ohmuro-Matsuyama
  • Hiroshi UedaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1461)

Abstract

There is a significant focus on detecting and assaying protein–protein interactions (PPIs) in biology and biotechnology. Protein-fragment complementation assay (PCA) is one of the most widely used methods to detect PPI by splitting the enzyme-coding or fluorescent protein-coding polypeptide, as well as Förster resonance energy transfer (FRET). Here, we describe a novel PPI assay FlimPIA (firefly luminescent intermediate-based protein–protein interaction assay) by a unique approach of splitting the two major catalytic steps (half reactions) of firefly luciferase (FLuc).

Key words

Protein–protein interaction Firefly luciferase Substrate channeling Reaction intermediate Luciferyl adenylate 

Notes

Acknowledgment

We thank B. Branchini for providing inspiration for the project. This project was supported by SENTAN, JST, Japan, and partly by the “Leave a Nest” Microtech Nichion award.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Chemical Science and Engineering, Graduate School of EngineeringKobe UniversityNada, KobeJapan
  2. 2.Laboratory for Chemistry and Life ScienceTokyo Institute of TechnologyMidori-ku, YokohamaJapan

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