Proteins are crucial for most cellular functions and typically participate in biological processes in concert with other proteins. Hence, identification of key protein players and characterization of protein-protein interactions (PPIs) are highly important. Owing to substantial advances in current biotechnology, a wide range of methods has been developed to dissect the PPI landscape. Given their popularity and power, the biomolecular fluorescence complementation (BiFC) assay, based on the reconstitution of a fluorescent protein in vivo, has emerged as the most popular protein-fragment complementation method in cellular biology over the past years.
My team has previously established BiFC for probing different binary protein interactions in live Drosophila. During my PhD work, I expanded the utility of the BiFC in mammalian live cells. In particular, I standardized the protocol of the BiFC analysis to investigate protein binding affinities, in an applicable and simple manner. This quantitative BiFC approach was used in a systematic analysis of HOX/PBX/MEIS interaction properties in live cells and revealed novel interaction interfaces in several human HOX proteins.
Furthermore, I applied the BiFC from low to high throughput PPI detection. Pairing sequence-verified human ORF collections with next generation sequencing, I participated to the conception of a powerful tool for performing a large-scale BiFC interaction screen in live cells. Benefited chiefly from this approach, a synoptic view of comprehensive HOX interactomes was delineated. My PhD work substantially contributes to the current limited knowledge on human HOX protein partners and provide a novel tool in the cell biology arsenal. Along with the contemporary development of proximity labeling methods and new Protein Complementation Assay design, in my side projects, I depicted and tested two more approaches for cell-based PPI detection. The first combining BiFC and BioID (proximity-dependent biotinylation identification) technonolgies, and allows to decipher the endogenous interactome of a protein complex. The other is inspired by tripartite split-GFP. association, a high-throughput BiFC-adapted method, enabling both binary and trinary PPI detection in live cells.
In summary, this PhD work demonstrates that the BiFC is a versatile and powerful tool to study PPIs in the live cellular context, on either small or large scale. More specially, under my continuous ongoing efforts, the BiFC assay has shown a potential to further combine with other methods and largely enriched the toolset by conferring enhanced or new functionalities.
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