Accurate and sensitive interactome profiling using a quantitative protein-fragment complementation assay

Area of Science:

• Molecular Biology
• Systems Biology
• Biochemistry

Background:

• Understanding protein-protein interactions (PPIs) is crucial for elucidating cellular mechanisms.
• Existing methods for mapping PPIs can be limited in precision and sensitivity.
• Genome-wide PPI mapping is essential for comprehensive cellular system analysis.

Purpose of the Study:

• To systematically probe protein-protein interactions using a novel quantitative assay.
• To demonstrate the precision and sensitivity of NanoLuc Binary Technology (NanoBiT) for PPI mapping.
• To establish a foundation for building accurate and comprehensive protein interaction maps.

Main Methods:

• Construction of genome-wide yeast strain libraries.
• Application of NanoLuc Binary Technology (NanoBiT), a quantitative protein-fragment complementation assay (PCA).
• Investigation of known PPIs and the interactome of selected proteins (Upf1, Cdc53, Met30).

Main Results:

• Demonstrated highly precise and sensitive mapping of protein-protein interactions using ratiometric NanoBiT measurements.
• Successfully probed interactions of well-documented PPIs and specific proteins like Upf1, Cdc53, and Met30.
• Validated NanoBiT as a robust tool for quantitative PPI analysis.

Conclusions:

• NanoBiT enables accurate and sensitive mapping of protein-protein interactions.
• This technology provides a foundation for assembling comprehensive protein interaction maps.
• NanoBiT facilitates deeper functional investigations of protein interactions within cellular systems.