Peptide tags are a key resource, introducing minimal change while enabling a consistent process to purify diverse proteins. However, peptide tags often provide minimal benefit post-purification. We previously designed SpyTag, forming an irreversible bond with its protein partner SpyCatcher. SpyTag provides an easy route to anchor, bridge or multimerize proteins. Here we establish Spy&Go, enabling protein purification using SpyTag. Through rational engineering we generated SpyDock, which captures SpyTag-fusions and allows efficient elution. Spy&Go enabled sensitive purification of SpyTag-fusions from Escherichia coli, giving superior purity than His-tag/nickel-nitrilotriacetic acid. Spy&Go allowed purification of mammalian-expressed, N-terminal, C-terminal or internal SpyTag. As an oligomerization toolbox, we established a panel of SpyCatcher-linked coiled coils, so SpyTag-fusions can be dimerized, trimerized, tetramerized, pentamerized, hexamerized or heptamerized. Assembling oligomers for Death Receptor 5 stimulation, we probed multivalency effects on cancer cell death. Spy&Go, combined with simple oligomerization, should have broad application for exploring multivalency in signaling.

肽标签是一种关键资源，它引入最小的变化，同时实现一致的过程来纯化不同的蛋白质。然而，肽标签通常在纯化后提供的益处微乎其微。我们之前设计了SpyTag，与其蛋白质伙伴SpyCatcher形成了不可逆转的联系。SpyTag 提供了一种锚定、桥接或多聚化蛋白质的简单途径。在这里，我们建立了 Spy&Go，使用 SpyTag 实现蛋白质纯化。通过合理的工程，我们生成了 SpyDock，它捕获 SpyTag 融合并允许有效的洗脱。Spy&Go 能够从大肠杆菌中灵敏地纯化 SpyTag 融合体，其纯度高于 His 标签/次氮基三乙酸镍。Spy&Go 允许纯化哺乳动物表达的 N 端、C 端或内部 SpyTag。作为寡聚工具箱，我们建立了一组 SpyCatcher 连接的卷曲线圈，因此 SpyTag 融合可以二聚、三聚、四聚、五聚、六聚或七聚。通过组装用于刺激死亡受体 5 的寡聚物，我们探讨了对癌细胞死亡的多价效应。Spy&Go 与简单的寡聚化相结合，应该在探索信号多价性方面具有广泛的应用。