A rapid, automation-friendly, cell-free and purification-free pipeline for binding and kinetic characterisation of de novo designed binders with high quality standardised per-design data

Abstract

De novo protein binders can now be generated far faster than they can be tested. Current validation routes - display selection, cell-free two-hybrid assays, or surface-based biophysics - require cell culture, colony picking, protein purification, target labeling or sensor immobilisation, take 2 to 8 weeks, and require additional methods for affinity and kinetics characterisation. Here we report an end-to-end, cell- and purification-free pipeline that goes from gene to binding affinity and kinetics in three days without genetically modified organisms, cell culture, purification, labelling or immobilisation. An automated, metric-driven loop of RFdiffusion, ProteinMPNN and AlphaFold2 initial-guess generates and filters designs, which are ranked by Boltz-2 ipSAE, assembled by Golden Gate cloning from linear DNA, expressed in a cell-free protein synthesis lysate as fluorescent protein fusions, and screened directly in the unpurified product by flow-induced dispersion analysis (FIDA). Against human growth hormone, 11 of 30 designs (37%) bound, and the four strongest had equilibrium dissociation constants of 0.93-9.98 nM with on- and off-rates resolved from the same cell-free material. Each sample run measures expression level, hydrodynamic radius, oligomeric and aggregation state alongside the binding readout, producing the standardised, per-design datasets that model training requires.