A Universal Expression Tag for Structural and Functional Studies of Proteins
A Universal Expression Tag for Structural and Functional Studies of Proteins
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用于蛋白质结构和功能研究的通用表达标签
DOI：
10.1002/cbic.201200045
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发表时间：
2012
期刊：
影响因子：
3.2
通讯作者：
Dötsch
中科院分区：
文献类型：
--
作者：
Rozenknop;Rogova;Tikole;Jaravine;Güntert;Dötsch
多肽和蛋白质的表达、分离和纯化是研究其结构和功能的关键步骤。近年来，许多报道表明，借助多种表达和溶解度标签（GST、MBP、NusA、thioredoxin、ubiquitin、SUMO、GB-1等），利用融合技术高效表达和分离多肽和蛋白质方面取得了很大进展。[1]在这些应用中，大多数融合标签是通过蛋白水解或化学裂解去除的，只有纯化后的肽/蛋白质才能用于进一步研究。然而，对于有聚集倾向的蛋白质和多肽，去除融合标签会导致严重的溶解度问题，阻碍了需要更高浓度的方法的研究，如核磁共振光谱或等温滴定量热法（ITC）。对于肽，由于它们相对于蛋白质的体积小，去除融合标签可能会导致关于它们的检测和纯化以及达到足够浓度的进一步问题。为了克服这种不利的情况，在研究过程中保持融合标签的附着可能是有益的，尽管这需要不改变蛋白质或肽的结构和功能特性。这种不可切割的可溶性增强标签（set）的概念是由Zhou等人引入的，用于通过溶液核磁共振研究“表现不佳”的蛋白质[2,3]。基于高可溶性小蛋白GB-1[4]的30多个蛋白质在结构上被成功研究然而，set的概念可以通过其他“通用”set得到更广泛的应用，并且可以通过在单个先导蛋白中结合表达、溶解度和稳定性增强标签来实现。在这项工作中，我们试图设计一种“通用”标签，它可以用于大多数生物物理和生化应用，并且可以在体外和体内研究肽/蛋白质及其相互作用。我们已经测试了设计的基于泛素的（Ubbased）标签在许多靶肽和蛋白质的结构和功能研究中的应用。与迄今为止最常用的“非相互作用”GB-1标签不同，Ub具有与几乎所有KD - > 0.3 mM的蛋白质非特异性相互作用的能力。具有n端His标签的野生型Ub已经在一些研究中成功地用作表达和溶解度标签这些融合结构的主要缺点是与SUMO或NusA标签[6]相比溶解度增强较低，并且融合结构在细菌中会自发降解[7]，[7]与Ub-fold识别和ElaD蛋白酶对UbÀGGÀX键的非特异性切割有关为了克服这些问题，我们重新设计了Ub序列，将含有His6标签和Ub的前导部分的等电点从7.1移到5.4，同时借助已有的数据提高了所得蛋白的热力学稳定性此外，我们将His6标签定位在Ub序列后面，以便使用Ub 5 ' -mRNA序列提供的增强表达水平（详见支持信息中的方案1A和图S1）。存在TEV切割位点的目的是在必要时通过反向纯化实现额外的蛋白质纯化步骤。选择TEV切割位点的原因是该蛋白酶在研究小组中的广泛使用，以及同时使用一系列蛋白酶抑制剂进行TEV切割的潜力。两个c端Gly…
The expression, isolation, and purification of peptides and proteins are the crucial initial steps for investigation of their structures and functions. In recent years, many reports have shown strong progress in efficient expression and isolation of peptides and proteins by use of fusion technology with the aid of a variety of expression and solubility tags: GST, MBP, NusA, thioredoxin, ubiquitin, SUMO, GB-1, etc.[1] In the majority of these applications the fusion tag is removed by proteolytic or chemical cleavage, and only the purified peptide/protein is used for further investigations. In cases of proteins and peptides that show a tendency to aggregate, however, the removal of the fusion tag can cause severe problems with solubility, preventing investigation with methods that require higher concentrations, such as NMR spectroscopy or isothermal titration calorimetry (ITC). For peptides, because of their small sizes relative to proteins, removal of a fusion tag can lead to further problems with regard to their detection and purification and to reaching sufficient concentrations. To overcome this unfavorable situation, it could be beneficial to keep the fusion tag attached during the investigations, although this requires that the structural and functional properties of the protein or peptide not be changed. The concept of such non-cleavable solubility-enhancement tags (SETs) for studies of “poorly behaved” proteins by solution NMR was introduced by Zhou et al.[2, 3] More than 30 proteins based on the small highly soluble protein GB-1 [4] were successfully studied structurally.[3] However, the idea of SETs can be significantly extended for broader usage with alternative “universal” SETs, and can be implemented by combining expression, solubility, and stability enhancement tags in a single leading protein. In this work we have tried to design a “universal” tag, which could be useful for most biophysical and biochemical applications and could be implemented both for in vitro and for in vivo studies of peptides/proteins and their interactions. We have tested the application of a designed ubiquitin-based (Ubbased) tag in structural and functional studies of a number of target peptides and proteins. In contrast with the “non-interacting” GB-1 tag most commonly used so far, Ub is known for its ability to interact nonspecifically with almost all proteins with KD> 0.3 mM. The wild-type Ub possessing an N-terminal His tag has already been successfully used as an expression and solubility tag in several studies.[5] The major drawbacks of these fusion constructs were lower solubility enhancement than with the SUMO or NusA tags [6] and a spontaneous degradation of the fused construct in bacteria,[7] which is connected to the Ub-fold recognition and nonspecific cleavage of the UbÀGGÀX bond by the ElaD protease.[8] In order to overcome these problems, we have redesigned the Ub sequence to shift the isoelectric point of the leading part containing a His6 tag and Ub from 7.1 to 5.4 and simultaneously to increase the thermodynamic stability of the resulting protein with the aid of reported data.[9] Additionally, we have positioned the His6 tag behind the Ub sequence in order to use the enhanced expression level provided by the Ub 5’-mRNA sequence (see Scheme 1A and Figure S1 in the Supporting Information for details). The purpose of the presence of a TEV cleavage site is to enable additional protein purification steps by reverse purification if necessary. The reasons for the choice of a TEV cleavage site are the broad usage of this protease in research groups and the potential to use a range of protease inhibitors simultaneously with TEV cleavage. The substitution of the two C-terminal Gly …
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影响因子：
5.6
作者：
Sgourakis NG;Patel MM;Garcia AE;Makhatadze GI;McCallum SA
通讯作者：
McCallum SA
通过 NMR 波谱探测 Y 受体 N 端结构域的特性
影响因子：
2.1
作者：
C. Zou;Sowmini Kumaran;R. Walser;O. Zerbe
通讯作者：
O. Zerbe
影响因子：
3.2
作者：
Lee, C;Lee, SG;Kim, BG
通讯作者：
Kim, BG
使用 Ubq-UIM 融合蛋白的温度诱导解折叠来实验测试蛋白质协同性的热力学模型。
影响因子：
2.9
作者：
Patel,MayankM;Sgourakis,NikolaosG;Garcia,AngelE;Makhatadze,GeorgeI
通讯作者：
Makhatadze,GeorgeI