Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), KTH Royal Institute of Technology, AlbaNova University Centre, Stockholm, Sweden.
Methods Mol Biol. 2021;2178:149-158. doi: 10.1007/978-1-0716-0775-6_12.
A positively charged protein domain, denoted Z, can be used as a general purification tag for purification of recombinantly produced target proteins by cation-exchange chromatography. The Z domain is constructed from the Protein A-derived Z-domain, and engineered to be highly charged, which allows selective capture on a cation exchanger at physiological pH values. Moreover, Z is selective also under denaturing conditions and can be used for purification of proteins solubilized from inclusion bodies. Z can then be used as a flexible linker to the cation-exchanger resin, and thereby allows solid-phase refolding of the target protein.Herein, protocols for purification of soluble Z-tagged fusion proteins , as well as for integrated purification and solid-phase refolding of insoluble fusion proteins , are described. In addition, a procedure for enzymatic tag removal and recovery of native target protein is outlined.
一个带正电荷的蛋白质结构域，称为 Z 结构域，可以作为一种通用的纯化标签，通过阳离子交换层析来纯化重组表达的目标蛋白。Z 结构域由蛋白 A 衍生的 Z 结构域构建而成，并经过工程改造使其带正电荷，这使得它可以在生理 pH 值下选择性地结合到阳离子交换剂上。此外，Z 结构域在变性条件下也具有选择性，可用于从包涵体中溶解的蛋白的纯化。Z 结构域可以用作与阳离子交换树脂的灵活连接子，从而允许目标蛋白的固相复性。本文描述了可溶性 Z 标签融合蛋白的纯化方案，以及不溶性融合蛋白的集成纯化和固相复性方案。此外，还概述了酶切标签去除和回收天然目标蛋白的过程。
