A high-throughput yeast two-hybrid protocol to determine virus-host protein interactions

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• The yeast two-hybrid (Y2H) system is crucial for identifying protein-protein interactions.
• Y2H has advanced understanding of viral protein structure, function, and virus-host interactions.
• Existing Y2H methods require optimization for large-scale screening.

Purpose of the Study:

• To present an optimized yeast two-hybrid (Y2H) protocol for high-throughput screening.
• To facilitate comprehensive analysis of intraviral and virus-host protein interactions.
• To enable automated and standardized screening of protein interaction networks.

Main Methods:

• Utilized yeast mating in a 96-well format for standardized liquid handling.
• Implemented a fluorescent reporter system for automated readout.
• Adapted the protocol for screening complex host cDNA libraries or arrayed protein pairs.

Main Results:

• The protocol enables highly standardized and fully automated high-throughput screening.
• Demonstrated ease of use, cost-effectiveness, and robotic handling capabilities.
• Facilitated extensive screening rounds for high coverage of protein-protein interactions.

Conclusions:

• This Y2H protocol represents an improved deep screening method for high-throughput assays.
• The system significantly expands the capacity to study protein interactions in virology and host-pathogen dynamics.
• Offers a powerful tool for comprehensive interactome mapping and biological discovery.