PA tag: A versatile protein tagging system using a super high affinity antibody against a dodecapeptide derived from human podoplanin
PA tag: A versatile protein tagging system using a super high affinity antibody against a dodecapeptide derived from human podoplanin
复制标题
DOI：
10.1016/j.pep.2014.01.009
复制
发表时间：
2014-03-01
影响因子：
1.6
通讯作者：
Takagi, Junichi
中科院分区：
文献类型：
--
作者：
Fujii, Yuki;Kaneko, Mika;Takagi, Junichi
基于肽的表位标记技术被广泛应用于几乎所有涉及目标蛋白生化表征的研究项目中，但并不是很多系统完全符合纯化目的。利用抗人podoplanin抗体nzi，我们构建了一个新的表位标签系统。z -1对被称为“PA标签”的十二肽具有异常高的亲和力，具有缓慢的解离动力学特征。由于其高亲和力，在稀释样品中，PA标记的蛋白质可以被固定的z -1树脂以近乎完全的方式捕获，并用游离PA肽溶液洗脱。这使得在哺乳动物细胞中表达的各种蛋白质，包括可溶性（神经匹林-1的外结构域片段）和膜（表皮生长因子受体）蛋白的一步高效纯化成为可能。肽结合抗体的温和再生条件确保了抗体树脂的重复使用，表明该系统具有成本效益。再加上其在免疫检测实验（即Western blotting和流式细胞术）中的出色表现，PA tag/ nz1系统将为许多生物医学研究项目提供促进蛋白质生产的巨大机会。(C) 2014爱思唯尔公司版权所有。
Peptide-based epitope tagging technology is universally used in nearly all kind of research projects that involve biochemical characterization of a target protein, but not many systems are fully compatible with purification purpose. By utilizing an anti-human podoplanin antibody NZ-I, we constructed a novel epitope tag system. NZ-1 possesses exceptionally high affinity toward a dodecapeptide dubbed "PA tag", with a characteristic slow dissociation kinetics. Because of its high affinity, PA-tagged proteins in a dilute sample can be captured by immobilized NZ-1 resin in a near complete fashion and eluted by a solution of free PA peptide. This enabled efficient one-step purification of various proteins including soluble (an ectodomain fragment of neuropilin-1) and membrane (epidermal growth factor receptor) proteins expressed in mammalian cells. Mild regeneration condition of the peptide-bound antibody ensures repeated use of the antibody resin, indicating a cost-efficient nature of the system. Together with its outstanding performance in the immunodetection experiments (i.e., Western blotting and flow cytometry), PA tag/NZ-1 system will offer a great chance to facilitate protein production in many biomedical research projects. (C) 2014 Elsevier Inc. All rights reserved.