首页> 外文OA文献 >A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli
【2h】

A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli

机译:大肠杆菌中重组基因表达常用调控启动子系统特性的比较分析

代理获取
本网站仅为用户提供外文OA文献查询和代理获取服务,本网站没有原文。下单后我们将采用程序或人工为您竭诚获取高质量的原文,但由于OA文献来源多样且变更频繁,仍可能出现获取不到、文献不完整或与标题不符等情况,如果获取不到我们将提供退款服务。请知悉。

摘要

Background: Production of recombinant proteins in bacteria for academic and commercial purposes is a well established field; however the outcomes of process developments for specific proteins are still often unpredictable. One reason is the limited understanding of the performance of expression cassettes relative to each other due to different genetic contexts. Here we report the results of a systematic study aiming at exclusively comparing commonly used regulator/promoter systems by standardizing the designs of the replicon backbones. Results: The vectors used in this study are based on either the RK2- or the pMB1-origin of replication and contain the regulator/promoter regions of XylS/Pm (wild-type), XylS/Pm ML1-17 (a Pm variant), LacI/P-T7lac, LacI/P-trc and AraC/P-BAD to control expression of different proteins with various origins. Generally and not unexpected high expression levels correlate with high replicon copy number and the LacI/P-T7lac system generates more transcript than all the four other cassettes. However, this transcriptional feature does not always lead to a correspondingly more efficient protein production, particularly if protein functionality is considered. In most cases the XylS/Pm ML1-17 and LacI/P-T7lac systems gave rise to the highest amounts of functional protein production, and the XylS/Pm ML1-17 is the most flexible in the sense that it does not require any specific features of the host. The AraC/P-BAD system is very good with respect to tightness, and a commonly used bioinformatics prediction tool (RBS calculator) suggested that it has the most translation-efficient UTR. Expression was also studied by flow cytometry in individual cells, and the results indicate that cell to cell heterogeneity is very relevant for understanding protein production at the population level. Conclusions: The choice of expression system needs to be evaluated for each specific case, but we believe that the standardized vectors developed for this study can be used to more easily identify the nature of case-specific bottlenecks. By then taking into account the relevant characteristics of each expression cassette it will be easier to make the best choice with respect to the goal of achieving high levels of protein expression in functional or nonfunctional form.
机译:背景:为学术和商业目的在细菌中生产重组蛋白是一个成熟的领域。但是,特定蛋白质的工艺开发结果通常仍然无法预测。原因之一是由于不同的遗传背景,对表达盒彼此之间的性能了解有限。在这里,我们报告了一项系统研究的结果,旨在通过标准化复制子主链的设计,专门比较常用的调节器/启动子系统。结果:本研究中使用的载体基于RK2或pMB1复制起点,并包含XylS / Pm(野生型),XylS / Pm ML1-17(Pm变体)的调节子/启动子区域。 ,LacI / P-T7lac,LacI / P-trc和AraC / P-BAD来控制具有各种起源的不同蛋白质的表达。通常且并非意料之外的是,高表达水平与高复制子拷贝数相关,并且LacI / P-T7lac系统比所有其他四个盒体产生更多的转录本。然而,这种转录特征并不总是导致相应更有效的蛋白质生产,特别是在考虑了蛋白质功能性的情况下。在大多数情况下,XylS / Pm ML1-17和LacI / P-T7lac系统产生的功能蛋白产量最高,而XylS / Pm ML1-17在不需要任何特定的意义上是最灵活的主机的功能。 AraC / P-BAD系统在密封性方面非常好,常用的生物信息学预测工具(RBS计算器)建议它具有最有效的翻译UTR。还通过流式细胞术研究了单个细胞中的表达,结果表明细胞间异质性对于了解群体水平的蛋白质生产非常相关。结论:需要针对每个特定病例评估表达系统的选择,但是我们认为为该研究开发的标准化载体可用于更轻松地鉴定特定病例瓶颈的性质。然后,通过考虑每个表达盒的相关特征,相对于实现功能性或非功能性形式的高水平蛋白表达的目标,更容易做出最佳选择。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
代理获取

客服邮箱:kefu@zhangqiaokeyan.com

京公网安备:11010802029741号 ICP备案号:京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有
  • 客服微信

  • 服务号