Authors
Ruiz Espi, D., Avelar Molina, G., Sonne Buch, O., Heyd, J., Unger, A., Winther Gormsen, L., Boldsen Lund, K., Sahni Ray, K., Williams, C., Hald, A., Jenkins, T. P., Rivera-de-Torre, E.
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.
Preprint server:
bioRxiv
The authors list and abstract were imported from bioRxiv on 21 Oct 2026.
Advertisement
Stats
- Recommendations n/a n/a positive of 0 vote(s)
- Views 12
- Comments 0