Novel, Versatile, and Tightly Regulated Expression System for Escherichia coli Strains
Novel, Versatile, and Tightly Regulated Expression System for Escherichia coli Strains
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DOI：
10.1128/aem.00413-10
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发表时间：
2010-08-01
影响因子：
4.4
通讯作者：
Miguez, Carlos B.
中科院分区：
文献类型：
--
作者：
Choi, Young J.;Morel, Lyne;Miguez, Carlos B.
利用恶臭假单胞菌（Pseudomonasputida）F1 cym和cmt操纵子的调控元件，以对异丙基苯甲酸（cumate）为诱导剂，在转录水平上控制目的基因的表达，构建了一种新的大肠杆菌基因表达系统。这种新的表达系统，被称为cumate基因开关，包括一个特定的表达载体，pNEW，它含有一个部分T5噬菌体启动子与假单胞菌为基础的合成操作子和cymR阻遏蛋白编码基因的组合，设计成在宿主菌株中组成型表达。转录的诱导依赖于添加外源诱导物（cumate），其对培养物无毒、水溶性且廉价。确定了表达系统的特性和潜力。使用流式细胞仪和补料分批发酵，我们已经表明，与新开发的cumate调节系统，（i）可以获得比pET更高的重组产物产量（异丙基-β-D-硫代吡喃半乳糖苷[IPTG]）诱导的表达系统，（ii）表达受到严格调控，（iii）与IPTG诱导的群体的双峰表达谱相反，cumate的添加快速导致完全诱导的和同质的蛋白质表达群体，（iv）表达可以通过改变cumate浓度来调节，和（v）cumate诱导的群体即使在诱导后8小时仍保持诱导和完全表达，导致高产量的靶蛋白。大肠杆菌菌株。
A novel tightly regulated gene expression system was developed for Escherichia coli by applying the regulatory elements of the Pseudomonas putida F1 cym and cmt operons to control target gene expression at the transcriptional level by using p-isopropylbenzoate (cumate) as an inducer. This novel expression system, referred to as the cumate gene switch, includes a specific expression vector, pNEW, that contains a partial T5 phage promoter combined with the Pseudomonas-based synthetic operator and the cymR repressor protein-encoding gene designed to express constitutively in the host strain. The induction of transcription relies on the addition of the exogenous inducer (cumate), which is nontoxic to the culture, water soluble, and inexpensive. The characteristics and potential of the expression system were determined. Using flow cytometry and fed-batch fermentations, we have shown that, with the newly developed cumate-regulated system, (i) higher recombinant product yields can be obtained than with the pET (isopropyl-beta-D-thiogalactopyranoside [IPTG])-induced expression system, (ii) expression is tightly regulated, (iii) addition of cumate quickly results in a fully induced and homogenous protein-expressing population in contrast to the bimodal expression profile of an IPTG-induced population, (iv) expression can be modulated by varying the cumate concentration, and (v) the cumate-induced population remains induced and fully expressing even at 8 h following induction, resulting in high yields of the target protein Furthermore, the cumate gene switch described in this article is applicable to a wide range of E. coli strains.