Engineering monomeric streptavidin and its ligands with infinite affinity in binding but reversibility in interaction
Engineering monomeric streptavidin and its ligands with infinite affinity in binding but reversibility in interaction
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DOI：
10.1002/prot.22446
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发表时间：
2009-11-01
影响因子：
2.9
通讯作者：
Wong, Sui-Lam
中科院分区：
文献类型：
--
作者：
Wu, Sau-Ching;Ng, Kenneth K. -S.;Wong, Sui-Lam
天然四聚体链亲和素通过超紧密结合（K-d类似于10（-13）到10(-14)M）捕获和固定生物素化分子。相比之下，工程单体链亲和素提供可逆结合（K-d类似于10(-7)M）。为了开发一种既具有天然链霉亲和素的固定化能力又具有链霉亲和素单体可逆相互作用活性的理想工程生物材料，通过分子模拟设计了一对工程生物材料。该系统由两个重组组分组成：一个是工程单体链亲和素M6，其在生物素结合位点附近有半胱氨酸残基（C118），另一个是含有生物素化标记的半胱氨酸。M6与生物素酸肽标签之间的相互作用通过两个阶段的过程（捕获和固定）来产生共价连接的复合物。生物素化在捕获阶段是必不可少的。一旦生物素化标签中的生物素片段被M6捕获，生物素化标签就可以折叠回并以肽标签中的生物素化赖氨酸为轴在复合物表面旋转，直到形成二硫键。因此，在生物素化标记中，半胱氨酸残基位于生物素残基两侧的不同位置，可以成功地与M6形成二硫键。分子间的二硫键在M6和含有蛋白质的标签之间形成，提供了对M6的固定能力。在还原剂和生物素存在的情况下，结合的配体可以解离。该系统有潜力扩展生物素-链亲和素技术，以开发可重复使用的生物传感器/蛋白质芯片和生物反应器。蛋白质2009;77:404 - 412。(C) 2009 Wiley-Liss, Inc。
Natural tetrameric streptavidin captures and immobilizes biotinylated molecules with ultra-tight binding (K-d similar to 10(-13) to 10(-14) M). In contrast, engineered monomeric streptavidin offers reversible binding (K-d similar to 10(-7) M). To develop an ideal engineered streptavidin which possesses both the immobilization capability of the natural streptavidin and the reversible interaction reactivity of the monomeric streptavidin, a pair of engineered biomaterials was designed through molecular modeling. This system consists of two recombinant components: an engineered monomeric streptavidin M6, which has a cysteine residue (C118) near the biotin binding site, and a cysteine containing biotinylation tag. Interactions between M6 and the biotinylate peptide tag go through a two-stage process (capture and immobilization) to generate a covalently linked complex. Biotinylation is essential in the capture stage. Once the biotin moiety in the biotinylated tag is captured by M6, the biotinylated tag can fold back and rotate on the surface of the complex with the biotinylated lysine in the peptide tag as the axis until the formation of a disulfide bond. Consequently, cysteine residue in different positions flanking the biotin residue in the biotinylation tag can successfully form a disulfide bond with M6. Intermolecular disulfide bond formation between M6 and the tag containing protein offers the immobilization capability to M6. In the presence of reducing agent and biotin, bound ligands can be dissociated. This system has the potential to extend the biotin-streptavidin technology to develop reusable biosensor/protein chips and bioreactors. Proteins 2009; 77:404-412. (C) 2009 Wiley-Liss, Inc.