Dual transcriptional-translational cascade permits cellular level tuneable expression control.
Dual transcriptional-translational cascade permits cellular level tuneable expression control.
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DOI：
10.1093/nar/gkv912
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发表时间：
2016-02-18
影响因子：
14.9
通讯作者：
Dixon N
中科院分区：
文献类型：
--
作者：
Morra R;Shankar J;Robinson CJ;Halliwell S;Butler L;Upton M;Hay S;Micklefield J;Dixon N
以小分子依赖的方式诱导基因表达的能力已经在靶标发现，功能阐明和生物生产中得到了许多应用。迄今为止，这些应用依赖于一套有限的基于蛋白质的控制机制，在转录起始水平上运作。核开关的发现、设计和重组为控制基因表达提供了另一种方法。在这里，我们报告了一种新的可调重组表达系统的发展和特征，称为RiboTite，它在转录和翻译水平上都起作用。利用标准诱导启动子和正交核开关，开发了多层模块化遗传控制电路来控制噬菌体T7 RNA聚合酶和重组基因的表达。该系统以许多常用的大肠杆菌表达系统为基准，显示出严格的基础控制，在细胞水平上基因表达的精确模拟可调性，在延长的生长期内对蛋白质产生率的剂量依赖性调节和增强的细胞活力。这种新系统扩大了用于重组蛋白生产的大肠杆菌表达系统的数量，并代表了在最广泛使用的表达系统之上的主要性能增强。
The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems.