A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli
A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli
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DOI：
10.1006/prep.2000.1363
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发表时间：
2001-02-01
影响因子：
1.6
通讯作者：
Tan, S
中科院分区：
文献类型：
--
作者：
Tan, S
为了促进多组分蛋白质复合物的研究，我开发了一种大肠杆菌表达系统，该系统可从单个质粒中共表达多达四种多肽。该系统的模块化性质使得能够将基因有效地亚克隆到多顺反子表达载体中的 4 个盒中的每一个中。多顺反子表达载体中存在的限制性位点允许亲和标记和未标记的复合物过表达，我证明了二元和三元复合物表达系统的成功使用，包括在大肠杆菌中重建VI-TG elonginC-elonginB复合物以及通过亲和和离子交换层析纯化复合物。这种多顺反子表达系统应该为多组分复合物的体外重建提供重要的替代方案。 (C) 2001 年学术出版社。
To facilitate studies of multicomponent protein complexes, I have developed an Escherichia coli expression system which coexpresses up to four polypeptides from a single plasmid, The modular nature of the system enables efficient subcloning of a gene into each of the 4 cassettes in the polycistronic expression vector. Restriction sites present in the polycistronic expression vector allow both affinity tagged and untagged complexes to be overexpressed, I demonstrate successful use of the expression system for binary and ternary complexes, including the reconstitution of the VI-TG elonginC-elonginB complex in E. coli and purification of the complex by affinity and ion-exchange chromatography, This polycistronic expression system should provide an important alternative to in vitro reconstitution of multicomponent complexes. (C) 2001 Academic Press.