Construction of a set Gateway-based destination vectors for high-throughput cloning and expression screening in Escherichia coli
Construction of a set Gateway-based destination vectors for high-throughput cloning and expression screening in Escherichia coli
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DOI：
10.1016/j.ab.2005.05.015
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发表时间：
2005-08-15
影响因子：
2.9
通讯作者：
Moras, D
中科院分区：
文献类型：
--
作者：
Busso, D;Delagoutte-Busso, B;Moras, D
我们在这里描述了一个10网关为基础的载体集适用于高通量克隆和表达重组蛋白在大肠杆菌的建设。质粒含有在T7启动子控制下产生重组蛋白所需的元件，并编码不同的N末端伴侣。由于载体组源自独特的骨架，因此融合配偶体对蛋白质表达和溶解度的影响的一致比较是容易进行的。最后，将编码六组氨酸标签的序列插入克隆的开放阅读框的C末端或N末端，从而提供选择六组氨酸标签位置用于进一步纯化的灵活性。为了测试我们的载体集的适用性，已经证明了两个枯草芽孢杆菌信号传导蛋白编码基因（SBGP编码E0508和E0511）的表达和溶解性特征以及六组氨酸标签可及性。(C)2005年爱思唯尔公司All rights reserved.
We describe here the construction of a 10-Gateway-based vector set applicable for high-throughput cloning and for expressing recombinant proteins in Escherichia coli. Plasmids bear elements required to produce recombinant proteins under control of the T7 promoter and encode different N-terminal partners. Since the vector set is derived from a unique backbone, a consistent comparison of the impact of fusion partner(s) on protein expression and solubility is easily amenable. Finally, a sequence encoding a six-histidine tag has been inserted to be in frame with the cloned open reading frame either at its C terminus or at the N terminus, giving the flexibility of choosing the six-histidine tag location for further purification. To test the applicability of our vector set, expression and solubility profile and six-histidine tag accessibility have been demonstrated for two Bacillus subtilis signaling proteins' encoding genes (SBGP codes E0508 and E0511). (C) 2005 Elsevier Inc. All rights reserved.