Abstract

De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37–43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing. A massively parallel computational and experimental approach for de novo designing and screening small hyperstable proteins targeting influenza haemagglutinin and botulinum neurotoxin B identifies new therapeutic candidates more robust than traditional antibody therapies. De novo protein design is a powerful tool for preparing small proteins with desired folds and functions. In this work, David Baker and colleagues report a combined computational and experimental approach to designing and screening folded mini-proteins, consisting of around 40 residues, to bind and target influenza haemagglutinin, a protein on the surface of the flu virus, and botulinum neurotoxin B, a cause of botulism. This high-throughput method produces binding proteins that are more stable and much smaller than traditional antibody therapies, that can be readily modulated and that elicit very little immune response. The optimal haemagglutinin binders show protection against influenza infection in vivo, illustrating the potential of this method for antiviral and other therapeutic applications.