Tabata Sanae, Kitago Yu, Fujii Yuki, Mihara Emiko, Tamura-Kawakami Keiko, Norioka Naoko, Takahashi Katsu, Kaneko Mika K, Kato Yukinari, Takagi Junichi
Institute for Protein Research, Osaka University, Suita, Osaka, 565-0871, Japan.
Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan.
Protein Expr Purif. 2018 Jul;147:94-99. doi: 10.1016/j.pep.2018.03.004. Epub 2018 Mar 14.
Peptide-based affinity tags are commonly used in recombinant production/purification of proteins, and are often preceded or followed by a protease recognition sequence to allow tag removal. We describe a rat monoclonal antibody 2H5 recognizing an undecapeptide tag called "eTev", which contains a recognition sequence for Tobacco Etch Virus (TEV) protease. In the crystal structure of 2H5-eTev complex, the long eTev peptide assumes compact α-helical conformation in the binding groove, exposing both ends to the solution. This architecture allowed us to connect eTev with another peptide tag called PA tag via linker sequence, ensuring the simultaneous access of two anti-tag antibodies. When this tandem double tag was attached at one end of various proteins, it enabled highly sensitive and protein-independent detection by sandwich ELISA. Utilizing this system during a rapid cell line screening, we succeeded in isolating stable cell clones expressing high level of mouse Wise protein.
基于肽的亲和标签常用于蛋白质的重组生产/纯化，并且通常在其前后带有蛋白酶识别序列，以便去除标签。我们描述了一种大鼠单克隆抗体2H5，它识别一种名为“eTev”的十一肽标签，该标签包含烟草蚀纹病毒（TEV）蛋白酶的识别序列。在2H5-eTev复合物的晶体结构中，长的eTev肽在结合槽中呈紧密的α螺旋构象，两端暴露于溶液中。这种结构使我们能够通过连接序列将eTev与另一种名为PA标签的肽标签连接起来，确保两种抗标签抗体能够同时接近。当这种串联双标签连接在各种蛋白质的一端时，它能够通过夹心ELISA进行高度灵敏且不依赖蛋白质的检测。在快速细胞系筛选过程中利用该系统，我们成功分离出了表达高水平小鼠Wise蛋白的稳定细胞克隆。
