High‐Throughput FRET Affinity Screening Technique (HTFAST) for Cell‐Free Expressed Binding Protein Characterization

The rapid engineering of binding proteins is increasingly driven by cell‐free, machine–learning–guided optimization. However, high‐throughput, quantitative characterization of binding affinity remains a major bottleneck, particularly for proteins expressed in cell‐free systems without purification. Here, we present High‐Throughput FRET Affinity Screening Technique (HTFAST) for rapid affinity characterization of binders expressed directly in crude E. coli cell‐free protein expression reactions. HTFAST leverages Förster resonance energy transfer (FRET) between cell‐free expressed fluorescent‐protein–fused binders and dye‐labeled antigens to enable real‐time, quantitative measurement of equilibrium dissociation constants. We systematically optimized fluorophore pairs and labeling parameters using the SpyTag003–SpyCatcher003 model system. HTFAST reliably quantified nanomolar binding affinities in crude lysates for the SpyTag003–SpyCatcher003 model system. It also resolved affinities spanning low‐nanomolar to hundreds‐of‐nanomolar for nanobodies against multiple antigens, including CD4, HER2, and HSA. We further benchmarked HTFAST against a panel of five SARS‐CoV‐2 RBD nanobodies, demonstrating HTFAST's ability to rank binders targeting the same antigen with affinities correlating strongly with reported BLI/SPR measurements. Finally, we demonstrate that both binding partners can be expressed directly in CFE, further streamlining screening workflows. Overall, HTFAST provides a scalable, quantitative, and cell‐free–compatible approach for high‐throughput affinity screening, well‐suited for DBTL protein engineering campaigns.