Parallelized identification of on- and off-target protein interactions

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Engineering

Background:

• Developing engineered proteins requires efficient methods to assess functionality and stability.
• Traditional methods struggle to screen large libraries of protein variants simultaneously.

Purpose of the Study:

• To develop and validate a high-throughput workflow for analyzing engineered protein variants.
• To enable simultaneous assessment of binding affinity and specificity for numerous protein interactions.

Main Methods:

• Displaying hundreds of engineered proteins on yeast cell surfaces.
• Utilizing flow cytometry for selection based on target molecule binding.
• Employing next-generation sequencing to quantify protein enrichment and determine binding characteristics.

Main Results:

• Successfully determined relative affinities and specificity profiles for thousands of protein interactions in parallel.
• Identified both intended interactions and unintended off-target binding events.
• Demonstrated the workflow's capability to screen engineered protein binders effectively.

Conclusions:

• This multiplex genetic selection and sequencing approach offers a general framework for screening engineered protein binders.
• The method is highly valuable for developing protein-based therapeutics by characterizing interactions and off-target effects.
• Facilitates the discovery and optimization of proteins for diverse biotechnological applications.