Improved validation of protein interactions using bicistronic BiFC (Bi2FC)

• 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
• ISSN: 0971-5894; 0974-0430
• DOI: 10.1007/s12298-024-01477-y

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.