Abiotic Synthetic Antibodies to Target a Specific Protein Domain and Inhibit Its Function.

Abstract

The Bacillus thuringiensis (Bt) Cry proteins are widely used in insect pest control. Despite their economic benefits, remaining concerns over potential ecological and health risks warrant their ongoing surveillance. Affinity reagents, most often antibodies, protein scaffolds, and aptamers, are the traditional tools used for protein binding and detection. We report a synthetic antibody (SA) alternative to traditional biological affinity reagents for binding Bt Cry proteins. Analysis of hotspots of the Bt Cry protein-insect midgut cadherin-like receptor complexes was used for the design of the SA. The SA was selected from a small focused library of hydrogel copolymers containing functional monomers complementary to key exposed hotspots of Bt Cry proteins. A directed chemical evolution identified a SA, APhe-NP23, with affinity and selectivity for Bt Cry1Ab/Ac proteins. The putative intermolecular polymer-protein interfaces were identified by the SA's uptake of Bt Cry1Ac pepsin hydrolysates, binding epitope mutation studies, and protein-protein inhibition studies of the toxin binding to its native insect receptor binding domains. The SA inhibitor binds to the same protein domains as the insect's cadherin-like receptors, Bt-R1 and SeCad1b. The SA binds rapidly to Bt Cry1Ab/Ac with high capacity, is pH-responsive, and is synthesized reproducibly. We believe that a hotspot-directed approach is general for creation of abiotic protein affinity reagents that target functional protein domains. Affinity ligands are typically high-information content biologicals. Their structure and function are determined from their amino acid or oligo sequence. In contract, the SA described in this work is a statistical copolymer that lacks sequence specificity. These results are an important contribution to the concept that randomness and biospecificity are not mutually exclusive.