Amplicon/Protein Bead Display enables quantitative in vitro biochemistry at scale

Scalable methods for producing and characterizing protein libraries are essential for generating standardized datasets needed to train models linking sequence to function. Here, we present Amplicon/Protein Bead Display (APB-Display), which expresses and purifies >100,000 protein variants in vitro in 1 day. APB-Display uses particle-templated emulsification to generate libraries of hydrogel beads that covalently display many copies of a given protein variant and its encoding DNA. By incubating beads with fluorescently-labeled ligands, sorting, and sequencing to generate titration curves (APB-TiteSeq), we simultaneously quantified expression levels and binding affinities ( K _d s) for >18,000 FLAG epitope variants interacting with M2 anti-FLAG antibody in 3 days, revealing chemistry-dependent epistasis between positions 4 and 5. Single-concentration binding measurements (APB-SortSeq) paired with neural network denoising further returned quantitative affinities for >88,000 variants. APB-Display requires only standard laboratory equipment and access to a FACS sorter, providing an accessible platform for quantitative in vitro biochemistry at scale.