High-Throughput FRET Affinity Screening Technique (HTFAST) For Cell-Free Expressed Binding Protein Characterization

Area of Science:

• Biotechnology
• Protein Engineering
• Biophysics

Background:

• Cell-free protein synthesis (CFPS) enables rapid engineering of high-affinity binding proteins.
• High-throughput characterization of binding affinity is a bottleneck, especially for unpurified proteins from CFPS.
• Machine learning guides optimization, necessitating efficient binding validation.

Purpose of the Study:

• To develop a high-throughput, quantitative method for binding affinity screening of cell-free expressed proteins.
• To enable rapid characterization of binding proteins directly in crude lysates without purification.
• To accelerate the development of next-generation binding proteins through streamlined screening.

Main Methods:

• Developed High-Throughput FRET Affinity Screening Technique (HTFAST) using Förster resonance energy transfer (FRET).
• Utilized fluorescent-protein-fused binders and dye-labeled antigens for real-time equilibrium dissociation constant measurement.
• Optimized fluorophore pairs and labeling parameters using the SpyTag003-SpyCatcher003 system.

Main Results:

• HTFAST reliably quantified nanomolar binding affinities in crude lysates.
• Validated the platform for nanobodies, including a CD4-binding nanobody (Nb457).
• Successfully benchmarked SARS-CoV-2 receptor-binding domain sdAbs, ranking their binding strengths.
• Demonstrated that both binding partners can be expressed directly in CFPS.

Conclusions:

• HTFAST offers a scalable, quantitative, and cell-free-compatible approach for high-throughput affinity screening.
• The method is well-suited for Design-Build-Test-Learn (DBTL) campaigns.
• Accelerates the development of high-affinity binding proteins for various applications.