Membrane protein expression and production: effects of polyhistidine tag length and position
Membrane protein expression and production: effects of polyhistidine tag length and position
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DOI：
10.1016/j.pep.2003.10.010
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发表时间：
2004-02-01
影响因子：
1.6
通讯作者：
Wiener, MC
中科院分区：
文献类型：
--
作者：
Mohanty, AK;Wiener, MC
多组氨酸标签使得能够通过固定化金属亲和层析（IMAC）容易地纯化蛋白质。纯化标签的类型和位置都可以显著影响蛋白质的性质，例如其表达水平、在溶液中的行为以及其形成合适样品（特别是用于X射线晶体学的合适晶体）的能力。我们系统地研究了多聚组氨酸标签的长度和位置对重组整合膜蛋白的表达和纯化相关的许多性质的影响。具体地，构建了修饰的大肠杆菌pET表达载体，其在亚克隆基因的N-或C-末端放置6-或10-组氨酸标签。急诊将大肠杆菌水通道蛋白AqpZ亚克隆到该载体中，并对其表达、纯化、溶液性质和产量进行了表征。这些研究表明：（1）所有载体产生相似的表达水平，（2）标签长度比标签位置对产量的影响更大，（3）标签长度和位置都不显著影响蛋白质的去污剂溶解，（4）标签的长度影响纯化蛋白质的寡聚化状态，和（5）去污剂溶解的蛋白质的标签长度和位置变化色谱行为。此外，在第二个位置的赖氨酸密码子AAA的取代，以前显示出对可溶性蛋白质表达水平有一定的影响，没有对AqpZ的生产有很大的影响。我们目前每升摇瓶培养物生产约12 mg纯化的AqpZ，并且已经获得了分辨率约为5 A的初步晶体。(C)2003年爱思唯尔公司All rights reserved.
Polyhistidine tags enable the facile purification of proteins by immobilized metal affinity chromatography (IMAC). Both the type and position of purification tags can affect significantly properties of a protein such as its expression level, behavior in solution, and its ability to form suitable samples (esp. suitable crystals for X-ray crystallography). We investigated systematically the effects of polyhistidine tag length and position on many properties related to expression and purification of recombinant integral membrane proteins. Specifically, modified Escherichia coli pET expression vectors were built that placed 6- or 10-histidine tags at the N- or C- termini of the subcloned gene. The E. coli water channel AqpZ was subcloned into this suite of vectors and its expression, purification, solution properties, and yield were characterized. These studies show that: (1) all vectors yield similar expression levels, (2) tag length has a greater effect than tag position upon yield, (3) neither tag length nor position affects significantly detergent solubilization of the protein, (4) the length of the tag affects the oligomerization state of the purified protein, and (5) the tag length and position change chromatographic behavior of the detergent-solubilized protein. In addition, substitution of the lysine codon AAA at the second position, previously shown to have some effect upon soluble protein expression levels, did not have a large effect on AqpZ production. We are currently producing approximately 12 mg of purified AqpZ per liter of shake-flask culture, and preliminary crystals that diffract to approximately 5 A resolution have been obtained. (C) 2003 Elsevier Inc. All rights reserved.