Analysis of Protein-Protein Interactions by Split Luciferase Complementation Assay

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Protein-protein interactions are critical in human disease pathogenesis.
• Understanding these interactions requires robust experimental tools.
• Developing methods to identify modulators of these interactions is essential.

Purpose of the Study:

• To describe and validate a split luciferase complementation (SLC) assay for discovering protein-protein interaction inhibitors.
• To provide detailed protocols for implementing the SLC assay.
• To demonstrate the utility of SLC using the NS2B-NS3 interaction as a model system.

Main Methods:

• Construction, expression, and purification of split luciferase fusion proteins.
• Assessment of different prey/bait pairs for optimal luminescence.
• Validation of SLC assay specificity and affinity.
• Step-by-step protocols for interaction assays, including competition binding and mutation analysis.

Main Results:

• Identification of optimal split luciferase fusion protein pairs for robust luminescence.
• Demonstration of SLC assay's ability to detect and quantify protein-protein interactions.
• Confirmation of assay specificity and affinity using various controls and mutations.
• Successful application of SLC to study the NS2B-NS3 interaction.

Conclusions:

• The described split luciferase complementation (SLC) method is a versatile and effective tool for identifying inhibitors of protein-protein interactions.
• The detailed protocols facilitate the application of SLC in diverse biological contexts, particularly for drug discovery.
• The NS2B-NS3 interaction serves as a validated model for the utility of SLC in studying essential viral protein interactions.