Here, we provide a protocol to generate synthetic nanobodies, known as sybodies, against any purified protein or protein complex within a 3-week period. Unlike methods that require animals for antibody generation, sybody selections are carried out entirely in vitro under controlled experimental conditions. This is particularly relevant for the generation of conformation-specific binders against labile membrane proteins or protein complexes and allows selections in the presence of non-covalent ligands. Sybodies are especially suited for cases where binder generation via immune libraries fails due to high sequence conservation, toxicity or insufficient stability of the target protein. The procedure entails a single round of ribosome display using the sybody libraries encoded by mRNA, followed by two rounds of phage display and binder identification by ELISA. The protocol is optimized to avoid undesired reduction in binder diversity and enrichment of non-specific binders to ensure the best possible selection outcome. Using the efficient fragment exchange (FX) cloning method, the sybody sequences are transferred from the phagemid to different expression vectors without the need to amplify them by PCR, which avoids unintentional shuffling of complementary determining regions. Using quantitative PCR (qPCR), the efficiency of each selection round is monitored to provide immediate feedback and guide troubleshooting. Our protocol can be carried out by any trained biochemist or molecular biologist using commercially available reagents and typically gives rise to 10-30 unique sybodies exhibiting binding affinities in the range of 500 pM-500 nM.

中文翻译：

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生成针对脆弱蛋白质的合成纳米抗体。

在这里，我们提供了一种在 3 周内针对任何纯化的蛋白质或蛋白质复合物生成合成纳米抗体（称为 sybodies）的协议。与需要动物产生抗体的方法不同，sybody 选择完全在受控实验条件下在体外进行。这与针对不稳定膜蛋白或蛋白质复合物的构象特异性结合剂的产生特别相关，并允许在存在非共价配体的情况下进行选择。Sybodies 特别适用于由于高序列保守性、毒性或靶蛋白稳定性不足而导致通过免疫文库生成结合剂失败的情况。该程序需要使用由 mRNA 编码的 sybody 文库进行一轮核糖体展示，然后通过 ELISA 进行两轮噬菌体展示和结合物鉴定。该协议经过优化，可避免粘合剂多样性的意外减少和非特异性粘合剂的富集，以确保获得最佳的选择结果。使用高效片段交换 (FX) 克隆方法，将 sybody 序列从噬菌粒转移到不同的表达载体，无需通过 PCR 扩增它们，避免了互补决定区的无意改组。使用定量 PCR (qPCR) 监控每一轮选择的效率，以提供即时反馈并指导故障排除。