Mertens N, Remaut E, Fiers W
Laboratory of Molecular Biology, University of Gent, Belgium.
Biotechnology (N Y). 1995 Feb;13(2):175-9. doi: 10.1038/nbt0295-175.
One of the more efficient systems for high-level expression of cloned genes in Escherichia coli makes use of a phage T7 late promoter whose activity depends on a regulatable transcription unit supplying the specific T7 RNA polymerase. Using various T7 RNA polymerase/T7 promoter-based vector host systems with differential control on expression of the T7 RNA polymerase, we document that leaky expression of the latter is responsible for the frequently observed loss of the culture's ability to express genes of interest. We further show that the inability to achieve detectable expression levels can be overcome by using a tightly repressed expression system. We describe a novel and efficient control system in which basal level expression of T7 RNA polymerase is attenuated by a series of tandemly arranged transcription terminators. The plasmids also incorporate the phage lambda-derived nutL/N protein antitermination function, allowing conditional reversion of attenuation upon induction. The applicability of the system is illustrated by the strictly regulatable, high-level production of several cytokines of human and murine origin.
在大肠杆菌中高效表达克隆基因的一种较为有效的系统利用了噬菌体T7晚期启动子，其活性取决于提供特异性T7 RNA聚合酶的可调控转录单元。使用各种基于T7 RNA聚合酶/T7启动子的载体宿主系统，对T7 RNA聚合酶的表达进行差异控制，我们证明后者的渗漏表达是导致培养物表达目的基因的能力经常丧失的原因。我们进一步表明，通过使用严格抑制的表达系统可以克服无法达到可检测表达水平的问题。我们描述了一种新型高效控制系统，其中T7 RNA聚合酶的基础水平表达通过一系列串联排列的转录终止子而减弱。这些质粒还整合了噬菌体λ来源的nutL/N蛋白抗终止功能，允许诱导后衰减的条件性逆转。该系统的适用性通过人和鼠源几种细胞因子的严格可调控的高水平生产得到了说明。
