Facile Display of Homomultivalent Proteins for In Vitro Selections

Low-affinity protein binders are emerging as valuable domains for therapeutic applications because of their higher specificity when presented in multivalent ligands that increase the overall strength and selectivity of receptor binding. De novo discovery of low-affinity binders would be enhanced by the large library sizes attainable with in vitro selection systems, but these platforms generally maximize recovery of high-affinity monovalent binders. Here, we present a facile technology that uses rolling circle amplification to create homomultivalent libraries. We show proof of principle of this approach in ribosome display with off-rate selections of a bivalent ligand against monovalent and bivalent targets, thereby demonstrating high enrichment (up to 166-fold) against a low-affinity target that is bivalent but not monovalent. This approach to homomultivalent library construction can be applied to any binder tolerant of N- and C-terminal fusions and provides a platform for performing in vitro display selections with controlled protein valency and orientation.