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Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System
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A screening system for identifying interacting proteins using biomolecular fluorescence complementation and

Honami Miyakura1, Mei Fukuda1, Hiroya Enomoto1

  • 1Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan.

Plos One
|May 14, 2021
PubMed
Summary

A novel screening system combining biomolecular fluorescence complementation and transposon gene trapping identifies novel protein interactions. This method successfully found new binding partners for p65 (RELA), a key NF-κB subunit.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Identifying protein-protein interactions is crucial for understanding cellular mechanisms.
  • Existing methods may not capture all relevant interactions, necessitating new approaches.

Purpose of the Study:

  • To develop and validate a novel screening system for identifying protein interactors.
  • To discover novel binding partners of the NF-κB subunit p65 (RELA).

Main Methods:

  • Established a screening system combining biomolecular fluorescence complementation (BiFC) and a transposon gene trap.
  • Created a bait strain expressing a fusion of monomeric Kusabira-Green (mKG) and a protein of interest.
  • Used a PiggyBac transposon to randomly fuse the remainder of mKG to endogenous genes for BiFC screening.

Main Results:

  • Screened for p65 (RELA) interactors, identifying PKM, HSP90AB1, ANXA2, HSPA8, and CACYBP.
  • Confirmed CACYBP as a novel p65 interactor using immunoprecipitation assays.
  • Demonstrated that CACYBP enhances NF-κB reporter activation, suggesting a role in NF-κB signaling.

Conclusions:

  • The developed BiFC-transposon gene trap system is effective for identifying novel protein interactions.
  • The findings reveal CACYBP as a novel regulator of NF-κB signaling.
  • This system offers a valuable tool for discovering previously unidentified interacting factors.