Picomolar affinity fibronectin domains engineered utilizing loop length diversity, recursive mutagenesis, and loop shuffling
Picomolar affinity fibronectin domains engineered utilizing loop length diversity, recursive mutagenesis, and loop shuffling
复制标题
DOI：
10.1016/j.jmb.2008.06.051
复制
发表时间：
2008-09-19
影响因子：
5.6
通讯作者：
Wittrup, K. Dane
中科院分区：
文献类型：
--
作者：
Hackel, Benjamin J.;Kapila, Atul;Wittrup, K. Dane
人纤连蛋白的第10个III型结构域（Fn3）已被证实是分子识别的有效支架。在当前的工作中，期望提高选择稳定的高亲和力Fn3结构域的稳健性。创建了Fn3的酵母表面展示文库，其中三个溶剂暴露的环在氨基酸组成和环长度方面多样化。通过荧光激活细胞分选筛选文库以分离溶菌酶的结合物。开发了亲和力成熟方案，以通过随机诱变以及诱变环的同源重组驱动的改组来快速且广泛地使克隆群体多样化。新的库和亲和力成熟方案相结合，以产生稳定的，单体的Fn3结构域与3 pM的溶菌酶的亲和力。二级亲和力成熟鉴定了稳定的1.1 pM结合剂，这是Fn3结构域报道的最高亲和力。除了扩展该支架的亲和力极限之外，结果还证明了在保持稳定性和单体状态的同时实现高亲和力结合的能力。该文库设计和亲和力成熟方案是高效的，利用2 × 10（7）克隆的初始多样性并仅筛选1 × 10（8）突变体（所有亲和力成熟文库的总和）。对中间群体的分析表明，环长度多样性、环改组和不同群体的递归诱变都是关键组成部分。(c)2008年由Elsevier Ltd.出版
The 10th type III domain of human fibronectin (Fn3) has been validated as an effective scaffold for molecular recognition. In the current work, it was desired to improve the robustness of selection of stable, high-affinity Fn3 domains. A yeast surface display library of Fn3 was created in which three solvent-exposed loops were diversified in terms of amino acid composition and loop length. The library was screened by fluorescence-activated cell sorting to isolate binders to lysozyme. An affinity maturation scheme was developed to rapidly and broadly diversify populations of clones by random mutagenesis as well as homologous recombination-driven shuffling of mutagenized loops. The novel library and affinity maturation scheme combined to yield stable, monomeric Fn3 domains with 3 pM affinity for lysozyme. A secondary affinity maturation identified a stable 1.1 pM binder, the highest affinity yet reported for an Fn3 domain. In addition to extension of the affinity limit for this scaffold, the results demonstrate the ability to achieve high-affinity binding while preserving stability and the monomeric state. This library design and affinity maturation scheme is highly efficient, utilizing an initial diversity of 2 x 10(7) clones and screening only 1 x 10(8) mutants (totaled over all affinity maturation libraries). Analysis of intermediate populations revealed that loop length diversity, loop shuffling, and recursive mutagenesis of diverse populations are all critical components. (c) 2008 Published by Elsevier Ltd.