Faculty of Basic Sciences, University of Medellin, Medellín, Colombia.
Grupo de Micología Médica y Experimental, Corporación para Investigaciones Biológicas (CIB), Medellín, Colombia.
BMC Res Notes. 2022 Mar 29;15(1):124. doi: 10.1186/s13104-022-06001-7.
To design and construct a new synthetic nanobody library using a structure-based approach that seeks to maintain high protein stability and increase the number of functional variants within the combinatorial space of mutations.
Synthetic nanobody (Nb) libraries are emerging as an attractive alternative to animal immunization for the selection of stable, high affinity Nbs. Two key features define a synthetic Nb library: framework selection and CDR design. We selected the universal VHH framework from the cAbBCII10 Nb. CDR1 and CDR2 were designed with the same fixed length as in cAbBCII10, while for CDR3 we chose a 14-long loop, which creates a convex binding site topology. Based on the analysis of the cAbBCII10 crystal structure, we carefully selected the positions to be randomized and tailored the codon usage at each position, keeping at particular places amino acids that guarantee stability, favoring properties like polarity at solvent-exposed positions and avoiding destabilizing amino acids. Gene synthesis and library construction were carried out by GenScript, using our own phagemid vector. The constructed library has an estimated size of 1.75 × 10. NGS showed that the amino acid diversity and frequency at each randomized position are the expected from the codon usage.
设计并构建一种基于结构的新型合成纳米抗体文库，旨在保持蛋白质的高度稳定性，并增加突变组合空间中的功能变体数量。
合成纳米抗体（Nb）文库作为一种替代动物免疫的方法，用于筛选稳定、高亲和力的 Nb，正逐渐受到关注。一个合成 Nb 文库有两个关键特征：框架选择和 CDR 设计。我们从 cAbBCII10 Nb 中选择了通用的 VHH 框架。CDR1 和 CDR2 的长度与 cAbBCII10 相同，而 CDR3 选择了 14 个氨基酸长的环，从而创造了一个凸形结合位点拓扑结构。基于 cAbBCII10 晶体结构的分析，我们仔细选择了要随机化的位置，并针对每个位置定制了密码子使用，在特定位置保留了保证稳定性的氨基酸，有利于在溶剂暴露位置具有极性等特性，并避免使用不稳定的氨基酸。基因合成和文库构建由 GenScript 完成，使用了我们自己的噬菌粒载体。构建的文库估计大小为 1.75×10。NGS 显示，每个随机化位置的氨基酸多样性和频率与密码子使用情况相符。
