Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
Angew Chem Int Ed Engl. 2017 Dec 22;56(52):16521-16525. doi: 10.1002/anie.201707623. Epub 2017 Dec 5.
SpyTag is a peptide that forms a spontaneous amide bond with its protein partner SpyCatcher. This protein superglue is a broadly useful tool for molecular assembly, locking together biological building blocks efficiently and irreversibly in diverse architectures. We initially developed SpyTag and SpyCatcher by rational design, through splitting a domain from a Gram-positive bacterial adhesin. In this work, we established a phage-display platform to select for specific amidation, leading to an order of magnitude acceleration for interaction of the SpyTag002 variant with the SpyCatcher002 variant. We show that the 002 pair bonds rapidly under a wide range of conditions and at either protein terminus. SpyCatcher002 was fused to an intimin derived from enterohemorrhagic Escherichia coli. SpyTag002 reaction enabled specific and covalent decoration of intimin for live cell fluorescent imaging of the dynamics of the bacterial outer membrane as cells divide.
SpyTag 是一个与它的蛋白质伴侣 SpyCatcher 自发形成酰胺键的肽。这种蛋白质超级胶水是一种非常有用的分子组装工具，可有效地将生物构建块锁定在各种结构中，且不可逆。我们最初通过合理设计，从革兰氏阳性细菌黏附素中拆分一个结构域，开发出 SpyTag 和 SpyCatcher。在这项工作中，我们建立了噬菌体展示平台来选择特定的酰胺化，导致 SpyTag002 变体与 SpyCatcher002 变体相互作用的速度提高了一个数量级。我们表明，002 对在广泛的条件下和蛋白质末端都能快速结合。SpyCatcher002 与来自产志贺毒素大肠杆菌的肠侵袭蛋白融合。SpyTag002 反应能够特异性和共价修饰肠侵袭蛋白，用于活细胞荧光成像，以观察细菌外膜在细胞分裂时的动力学。
