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

• Published in: ACS synthetic biology 2016-12, Vol.5 (12), p.1348-1354
• Main Authors: Carlin, Kevin B, Cruz-Teran, Carlos A, Kumar, Jay Prakash, Gomes, Catherina, Rao, Balaji M
• Format: Article
• Language: English
• Subjects: Archaeal Proteins - genetics; Archaeal Proteins - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Mutagenesis; Peptide Library; Plasmids - genetics; Protein Conformation; Protein Engineering; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Yeasts - genetics; Yeasts - metabolism
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.6b00034

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 K D ∼ 1.3 μM and 250 nM produced a bivalent binder with apparent K D ∼ 2 nM. Furthermore, the bivalent protein retained thermal stability (T M = 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.