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首页> 外文期刊>Biotechnology Progress >Coupling the T7 A1 promoter to the runaway-replication vector as anefficient method for stringent control and high-level expression of lacZ
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Coupling the T7 A1 promoter to the runaway-replication vector as anefficient method for stringent control and high-level expression of lacZ

机译：将T7 A1启动子与失控复制载体偶联作为lacZ严格控制和高水平表达的有效方法

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摘要

An expression vector characterized by tight regulation and high expression of cloned genes appears to be indispensable for the engineering need. To achieve this goal, in association with lacI the T7 A1 promoter containing two synthetic lac operators was constructed into a runaway-replication vector. To further examine this vector system, lacZ was subcloned and placed under the control of the T7 A1 promoter on the plasmid. With the application of the thermal induction alone, the Escherichia coli strain harboring the recombinant plasmid was able to produce 15,000 Miller units of beta -galactosidase, while it yielded the recombinant protein with 45,000-50,000 Miller units upon both thermal and chemical induction. In sharp contrast, only 60-90 Miller units of beta -galactosidase was obtained for the cell at an uninduced state. As a result, the production yield of beta -galactosidase over the background level is amplified approximately 170-fold by thermal induction and 500-fold by thermal and chemical induction. To produce the recombinant protein on a large scale, an approach by connecting two fermenters in series was newly developed. By applying the three-stage temperature shift in this dual fermenter system, 55,000 Miller units of beta -galactosidase was obtained. Overall, it shows the potential use of the vector system developed here for its tight control and high production of recombinant proteins.
机译：对于工程需要而言，以严格调控和克隆基因的高表达为特征的表达载体似乎是必不可少的。为了实现该目标，与lacI结合，将包含两个合成lac操纵子的T7 A1启动子构建为失控复制载体。为了进一步检查该载体系统，将lacZ亚克隆并置于质粒上T7 A1启动子的控制下。仅通过热诱导的应用，带有重组质粒的大肠杆菌菌株能够产生15,000 Miller单位的β-半乳糖苷酶，而通过热诱导和化学诱导均产生具有45,000-50,000 Miller单位的重组蛋白。与之形成鲜明对比的是，未诱导状态的细胞仅获得60-90 Miller单位的β-半乳糖苷酶。结果，超过背景水平的β-半乳糖苷酶的产量通过热诱导被扩增了约170倍，并且通过热和化学诱导被扩增了约500倍。为了大规模生产重组蛋白，新开发了通过串联连接两个发酵罐的方法。通过在该双重发酵罐系统中施加三阶段温度变化，获得了55,000密勒单位的β-半乳糖苷酶。总体而言，它显示了此处开发的载体系统在严格控制和高产量重组蛋白方面的潜在用途。

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