A monovalent streptavidin with a single femtomolar biotin binding site
A monovalent streptavidin with a single femtomolar biotin binding site
复制标题
DOI：
10.1038/nmeth861
复制
发表时间：
2006-04-01
期刊：
影响因子：
48
通讯作者：
Ting, AY
中科院分区：
文献类型：
--
作者：
Howarth, M;Chinnapen, DJF;Ting, AY
链霉亲和素和亲和素被广泛使用，因为它们与生物素结合有显著的亲和力，但它们是四聚体，这扰乱了它们的许多应用。由于结合部位的一部分来自相邻的亚基，使任一种蛋白质单体的亲和力降低至少10(4)倍。在这里，我们设计了一个链霉亲和素四聚体，它只有一个功能生物素结合亚基，保持了野生型链霉亲和素的亲和力、脱落率和热稳定性。在变性剂中，我们混合了一个链霉亲和素变异体，其中包含三个突变，以3：1的比例阻止生物素与野生型链霉亲和素结合。然后通过复性和亲和镍纯化得到单价链霉亲和素。同样，我们提纯了具有两个或三个生物素结合亚基的已定义的四聚体。用单价链霉亲和素标记位点特异性的神经连接素-1可以在没有交联物的情况下稳定地追踪神经连接素-1，而野生型链霉亲和素聚集神经连接素-1并扰乱突触前接触。单价链霉亲和素有望在生物分子标记、单粒子示踪和纳米技术中得到广泛应用。
Streptavidin and avidin are used ubiquitously because of the remarkable affinity of their biotin binding, but they are tetramers, which disrupts many of their applications. Making either protein monomeric reduces affinity by at least 10(4)-fold because part of the binding site comes from a neighboring subunit. Here we engineered a streptavidin tetramer with only one functional biotin binding subunit that retained the affinity, off rate and thermostabitity of wild-type streptavidin. In denaturant, we mixed a streptavidin variant containing three mutations that block biotin binding with wild-type streptavidin in a 3:1 ratio. Then we generated monovalent streptavidin by refolding and nickel-affinity purification. Similarly, we purified defined tetramers with two or three biotin binding subunits. Labeling of site-specifically biotinytated neuroligin-1 with monovalent streptavidin allowed stable neuroligin-1 tracking without cross-Linking, whereas wild-type streptavidin aggregated neuroligin-1 and disrupted presynaptic contacts. Monovalent streptavidin should find general application in biomolecule labeling, single-particle tracking and nanotechnology.