Combinatorial Pairwise Assembly Efficiently Generates High Affinity Binders and Enables a "Mix-and-Read" Detection Scheme.

We show that a combinatorial library constructed by random pairwise assembly of low affinity binders can efficiently generate binders with increased affinity. Such a library based on the Sso7d scaffold, from a pool of low affinity binders subjected to random mutagenesis, contained putative high affinity clones for a model target (lysozyme) at higher frequency than a library of monovalent mutants generated by random mutagenesis alone. Increased binding affinity was due to intramolecular avidity generated by linking binders targeting nonoverlapping epitopes; individual binders of KD ∼ 1.3 µM and 250 nM produced a bivalent binder with apparent KD ∼ 2 nM. Furthermore, the bivalent protein retained thermal stability (TM = 84.5 °C) and high recombinant expression yields in E. coli. Finally, when binders comprising the bivalent protein are fused to two of the three fragments of tripartite split-green fluorescent protein (GFP), target-dependent reconstitution of fluorescence occurs, thereby enabling a "mix-and-read" assay for target quantification.