A cell-based protein-protein interaction method using a permuted luciferase reporter.

Area of Science:

• Biochemistry
• Molecular Biology
• Assay Development

Background:

• Protein-protein interactions (PPIs) are crucial for cellular functions.
• Existing PPI assay methods have limitations in sensitivity and applicability.
• Developing novel, versatile assays for PPIs is essential for biological research.

Purpose of the Study:

• To develop and validate a novel cell-based assay for detecting protein-protein interactions.
• To demonstrate the assay's utility across different classes of receptors.

Main Methods:

• A split luciferase reporter system was engineered, incorporating a Tobacco Etch Virus (TEV) protease cleavage site.
• Protein A was fused to one part of the split luciferase and the TEV protease sequence.
• Protein B was fused to the other part of the split luciferase, enabling interaction-dependent activation.

Main Results:

• The assay demonstrated successful detection of protein-protein interactions.
• Luciferase activity was restored upon specific protein interactions and subsequent TEV protease cleavage.
• The assay was validated for ligand-induced interactions involving G-protein coupled receptors, receptor tyrosine kinases, and nuclear hormone receptors.

Conclusions:

• The developed cell-based assay provides a sensitive and versatile method for studying protein-protein interactions.
• This novel assay is applicable to various signaling pathways and receptor families.
• The method offers a valuable tool for drug discovery and understanding cellular mechanisms.