A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli.
A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli.
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DOI：
10.1186/1475-2859-12-26
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发表时间：
2013-03-18
影响因子：
6.4
通讯作者：
Valla S
中科院分区：
文献类型：
--
作者：
Balzer S;Kucharova V;Megerle J;Lale R;Brautaset T;Valla S
在细菌中生产用于学术和商业目的的重组蛋白是一个成熟的领域；然而，特定蛋白质的工艺发展结果往往仍然是不可预测的。一个原因是由于不同的遗传背景，对表达磁带相对于彼此的表现的理解有限。在这里，我们报告了一项系统研究的结果，该研究旨在通过标准化复制子主干的设计来专门比较常用的调控子/启动子系统。本研究中使用的载体基于RK2-或pMB1-复制起源，并包含XylS/Pm（野生型），XylS/Pm ML1-17 （Pm变体），LacI/PT7lac， LacI/Ptrc和AraC/PBAD的调节/启动子区域，以控制不同来源的不同蛋白质的表达。通常情况下，高表达水平与高复制子拷贝数相关，并且LacI/PT7lac系统比所有其他四种磁带产生更多的转录本。然而，这种转录特征并不总是导致相应的更有效的蛋白质生产，特别是如果考虑到蛋白质的功能。在大多数情况下，XylS/Pm ML1-17和LacI/PT7lac系统产生最多的功能蛋白，XylS/Pm ML1-17是最灵活的，因为它不需要宿主的任何特定特征。AraC/PBAD系统在紧密性方面非常好，常用的生物信息学预测工具（RBS计算器）表明它具有最有效的翻译UTR。流式细胞术还研究了单个细胞的表达，结果表明细胞间的异质性与在群体水平上理解蛋白质产生非常相关。表达系统的选择需要针对每个特定病例进行评估，但我们相信为本研究开发的标准化载体可以更容易地用于识别特定病例瓶颈的性质。然后，考虑到每个表达盒的相关特征，将更容易做出最佳选择，以实现高水平的功能或非功能形式的蛋白质表达。
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. 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/PT7lac, LacI/Ptrc and AraC/PBAD 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/PT7lac 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/PT7lac 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/PBAD 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. 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 non-functional form.
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影响因子：
5.7
作者：
Vee Aune TE;Bakke I;Drabløs F;Lale R;Brautaset T;Valla S
通讯作者：
Valla S
影响因子：
3.5
作者：
Cormack, BP;Valdivia, RH;Falkow, S
通讯作者：
Falkow, S
影响因子：
2.7
作者：
Giacalone, MJ;Gentile, AM;Surber, MW
通讯作者：
Surber, MW
影响因子：
4.4
作者：
Bakke, Ingrid;Berg, Laila;Valla, Svein
通讯作者：
Valla, Svein
影响因子：
4.4
作者：
Choi, Young J.;Morel, Lyne;Miguez, Carlos B.
通讯作者：
Miguez, Carlos B.