Combinatorial Pairwise Assembly Efficiently Generates High Affinity Binders and Enables a "Mix-and-Read" Detection Scheme
Combinatorial Pairwise Assembly Efficiently Generates High Affinity Binders and Enables a "Mix-and-Read" Detection Scheme
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DOI：
10.1021/acssynbio.6b00034
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发表时间：
2016-12-01
影响因子：
4.7
通讯作者：
Rao, Balaji M.
中科院分区：
文献类型：
--
作者：
Carlin, Kevin B.;Cruz-Teran, Carlos A.;Rao, Balaji M.
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 similar to 1.3 mu M and 250 nM produced a bivalent binder with apparent K-D similar to 2 nM. Furthermore, the bivalent protein retained thermal stability (T-M = 84.S degrees 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.
