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

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• 作者：Kevin B. Carlin ; Carlos A. Cruz-Teran ; Jay Prakash Kumar ; Catherina Gomes ; Balaji M. Rao
• 刊名：ACS Synthetic Biology
• 出版年：2016
• 出版时间：December 16, 2016
• 年：2016
• 卷：5
• 期：12
• 页码：1348-1354
• 全文大小：465K
• ISSN：2161-5063

文摘

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.