The isolation of nanobodies from pre-immune libraries by means of biopanning is a straightforward process. Nevertheless, the recovered candidates often require optimization to improve some of their biophysical characteristics. In principle, CDRs are not mutated because they are likely to be part of the antibody paratope, but in this work, we describe a mutagenesis strategy that specifically addresses CDR1. Its sequence was identified as an instability hot spot by the PROSS program, and the available structural information indicated that four CDR1 residues bound directly to the antigen. We therefore modified the loop flexibility with the addition of an extra glycine rather than by mutating single amino acids. This approach significantly increased the nanobody yields but traded-off with moderate affinity loss. Accurate modeling coupled with atomistic molecular dynamics simulations enabled the modifications induced by the glycine insertion and the rationale behind the engineering design to be described in detail.

中文翻译：

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CDR1 组合物可影响纳米抗体重组表达产量

通过生物淘选从免疫前文库中分离纳米抗体是一个简单的过程。然而，恢复的候选者通常需要优化以改善它们的一些生物物理特性。原则上，CDR 不会发生突变，因为它们可能是抗体互补位的一部分，但在这项工作中，我们描述了一种专门针对 CDR1 的诱变策略。其序列被 PROSS 程序鉴定为不稳定热点，可用的结构信息表明四个 CDR1 残基直接与抗原结合。因此，我们通过添加额外的甘氨酸而不是通过突变单个氨基酸来修改环的灵活性。这种方法显着提高了纳米抗体的产量，但以适度的亲和力损失为代价。