A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production.
A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production.
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DOI：
10.1186/1472-6750-6-12
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发表时间：
2006-03-01
影响因子：
3.5
通讯作者：
Bottomley SP
中科院分区：
文献类型：
--
作者：
Cabrita LD;Dai W;Bottomley SP
在过去的几年里，自动化和手动高通量蛋白表达和纯化已成为一种可访问的手段，快速筛选和生产可溶性蛋白的结构和功能的研究。然而，许多编码不同可溶性标签的商业载体对于每个载体需要不同的克隆和纯化步骤，这大大减慢了表达筛选。我们开发了一套E.具有不同可溶性标签的大肠杆菌表达载体，其允许从单个PCR产物平行克隆，并且可以使用相同的方案纯化。E.大肠杆菌表达载体，编码六组氨酸标签或三种最常用的可溶性标签（GST、MBP、NusA），并且全部具有N-末端六组氨酸序列。结果是双重的：His-标签有助于通过固定化金属亲和层析进行纯化，而融合结构域除了提供任选的纯化步骤之外，还主要在表达期间充当溶解性助剂。我们还在可溶性标签结构域之后掺入了TEV识别序列，其允许融合蛋白的高度特异性切割（使用TEV蛋白酶）以产生天然蛋白。这些载体也被设计用于非连接依赖性克隆，并且它们具有高水平表达的T7启动子，其适合于自诱导。为了验证我们的载体系统，我们克隆了四个不同的基因，也有一个基因到所有四个载体，并使用小规模表达和纯化技术。我们证明，载体能够高水平的表达和新蛋白的有效筛选，可以很容易地实现在实验室水平。其结果是一组四个合理设计的载体，可用于在实验室中的目的蛋白的流线型克隆，表达和纯化，并具有适应于高通量筛选的潜力。
In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol. The set of E. coli expression vectors, encode for either a hexa-histidine tag or the three most commonly used solubility tags (GST, MBP, NusA) and all with an N-terminal hexa-histidine sequence. The result is two-fold: the His-tag facilitates purification by immobilised metal affinity chromatography, whilst the fusion domains act primarily as solubility aids during expression, in addition to providing an optional purification step. We have also incorporated a TEV recognition sequence following the solubility tag domain, which allows for highly specific cleavage (using TEV protease) of the fusion protein to yield native protein. These vectors are also designed for ligation-independent cloning and they possess a high-level expressing T7 promoter, which is suitable for auto-induction. To validate our vector system, we have cloned four different genes and also one gene into all four vectors and used small-scale expression and purification techniques. We demonstrate that the vectors are capable of high levels of expression and that efficient screening of new proteins can be readily achieved at the laboratory level. The result is a set of four rationally designed vectors, which can be used for streamlined cloning, expression and purification of target proteins in the laboratory and have the potential for being adaptable to a high-throughput screening.
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影响因子：
14.9
作者：
ASLANIDIS, C;DEJONG, PJ
通讯作者：
DEJONG, PJ
DOI：
10.1016/j.bbrc.2004.07.189
发表时间：
2004-09-24
影响因子：
3.1
作者：
De Marco, V;Stier, G;de Marco, A
通讯作者：
de Marco, A
影响因子：
3.5
作者：
DIGUAN, C;LI, P;INOUYE, H
通讯作者：
INOUYE, H
影响因子：
8
作者：
Ke, A;Wolberger, C
通讯作者：
Wolberger, C
DOI：
10.1023/b:jsfg.0000031965.37625.0e
发表时间：
2004-01-01
期刊：
Journal of Structural and Functional Genomics
影响因子：
--
作者：
Woestenenk, Esmeralda A.;Hammarstrom, Martin;Berglund, Helena
通讯作者：
Berglund, Helena