Improved validation of protein interactions using bicistronic BiFC (Bi2FC)

Refolding based Bimolecular Fluorescence Complementation (BiFC) has emerged as an important in vivo technique to identify protein interactions. Significant improvements have been made to enhance the detection capacities of BiFC, however less attention has been paid to the detection of expression lev...

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Bibliographic Details
Published in:Physiology and molecular biology of plants 2024-07, Vol.30 (7), p.1047-1054
Main Authors: Sivakumar, Prakash, Vaishnavi, Vijayaraj, Gayatri, Kothuri, Satheesh, Gayathri R., Siddiqi, Imran
Format: Article
Language:English
Subjects:
Biological and Medical Physics
Biomedical and Life Sciences
Biophysics
Cell Biology
Fluorescence
Identification methods
In vivo methods and tests
Life Sciences
Plant Physiology
Plant Sciences
Protein folding
Protein interaction
Proteins
Robust control
Short Communication
Yeasts
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Summary:Refolding based Bimolecular Fluorescence Complementation (BiFC) has emerged as an important in vivo technique to identify protein interactions. Significant improvements have been made to enhance the detection capacities of BiFC, however less attention has been paid to the detection of expression levels of proteins. Here we demonstrate development and validation of an improved method to identify protein interactions that incorporates an expression control based on bicistronic expression of the protein of interest and a fluorescent protein separated by a self-cleaving peptide. This method gives robust identification of positive interactions and more reliably identifies absence of interactions. We also show an earlier identified non-interacting pair in yeast two-hybrid (Y2H) to be interacting in vivo.
ISSN:0971-5894
0974-0430
DOI:10.1007/s12298-024-01477-y