High-level production of aquaporin Z in Escherichia coli using maltose-binding protein/polyhistidine dual-affinity tag fusion system
High-level production of aquaporin Z in Escherichia coli using maltose-binding protein/polyhistidine dual-affinity tag fusion system
复制标题
使用麦芽糖结合蛋白/多组氨酸双亲和标签融合系统在大肠杆菌中高水平生产水通道蛋白 Z
DOI：
10.1016/j.procbio.2016.02.002
复制
发表时间：
2016-05-01
影响因子：
4.4
通讯作者：
Xu, Zhinan
中科院分区：
文献类型：
--
作者：
Hang, Baojian;Pan, Jianfeng;Xu, Zhinan
水通道蛋白Z（AqpZ）是属于主要内在蛋白（MIP）家族的一种水通道蛋白，存在于大肠杆菌的膜中。由于其高透水性和低活化能，AqpZ是开发仿生水过滤技术的潜在候选者。在本研究中，膜相关AqpZ在E.利用麦芽糖结合蛋白（MBP）/多组氨酸（HIS）双亲和标签融合系统，构建了一个重组质粒pET-MBP-HIS。系统研究了不同宿主菌和表达条件对MBP-AqpZ-HIS融合蛋白表达的影响。此外，开发了一种通过亲和层析纯化AqpZ-HIS的有效方案，并且从11 μ l培养物中获得了约15-20 mg功能性AqpZ-HIS，其比水转运活性约为2 × 10（-14）cm（3）s（-1）单体（-1）。十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳（PAGE）和色氨酸荧光发射分析表明，纯化的AqpZ-HIS以同源四聚体的形式组装在一起，具有较高的热稳定性，从而表明AqpZ的正确折叠。结果表明，MBP/HIS双亲和标签系统在大肠杆菌MIP通道蛋白的过表达、膜靶向和纯化方面是高效的。杆菌(C)2016爱思唯尔有限公司版权所有
Aquaporin Z (AqpZ) is a water channel protein belonging to the major intrinsic protein (MIP) family and is found in the membrane of Escherichia coli. Due to its high water permeability and low activation energy, AqpZ is a potential candidate for developing biomimetic water filtration technologies. In the present study, membrane-associated AqpZ was expressed in E. coli using the maltose-binding protein (MBP)/polyhistidine (HIS) dual-affinity tag fusion system. The effects of different host strains and the expression conditions on the production of the fusion protein MBP-AqpZ-HIS were systematically studied. Furthermore, an efficient protocol was developed to purify AqpZ-HIS via affinity chromatography, and approximately 15-20 mg of functional AqpZ-HIS was obtained from 11 of culture with a specific water transport activity of around 2 x 10(-14) cm(3) s(-1) monomer(-1). The related sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and tryptophan fluorescence emission analyses indicated that purified AqpZ-HIS assembled as a homotetramer with high thermostability, in turn indicating the correct folding of AqpZ. Our results showed that the MBP/HIS dual-affinity tag system was highly efficient for the overexpression, membrane targeting, and purification of MIP channel proteins in E. coli. (C) 2016 Elsevier Ltd. All rights reserved.