Z：一种用于选择性离子交换回收的纯化标签。

Z: A Purification Tag for Selective Ion-Exchange Recovery.

机构信息

Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), KTH Royal Institute of Technology, AlbaNova University Centre, Stockholm, Sweden.

出版信息

Methods Mol Biol. 2021;2178:149-158. doi: 10.1007/978-1-0716-0775-6_12.

DOI:10.1007/978-1-0716-0775-6_12

PMID:33128749

Abstract

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.

摘要

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。