Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system.
Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system.
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DOI：
10.1186/1754-1611-4-9
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发表时间：
2010-07-01
影响因子：
5.6
通讯作者：
Noireaux V
中科院分区：
文献类型：
--
作者：
Shin J;Noireaux V
利用基于噬菌体转录的大肠杆菌无细胞表达系统，可以在数小时内合成大量重组蛋白。这些细胞质提取物用于许多需要大规模蛋白质生产的应用，例如蛋白质组学和高通量技术。近年来，无细胞系统也被用来设计复杂的信息过程。然而，这些工作受到当前可用的无细胞系统的限制，这些系统不能很好地适应这些类型的研究。特别是，尚未提出提高无细胞反应中mRNA失活率和蛋白质降解率的方法。具有有趣动态的体外信息过程的构建需要 mRNA 和蛋白质合成（源）以及 mRNA 失活和蛋白质降解（汇）之间的平衡。两项定量研究旨在表征和提高整体 mRNA 失活率，并加速内源性 RNA 聚合酶和 Sigma 因子 70 驱动的大肠杆菌无细胞表达系统中合成蛋白质的降解。大肠杆菌 mRNA 干扰酶 MazF 用于提高和调整萤火虫荧光素酶 (Luc) 和增强型绿色荧光蛋白 (eGFP) 的 mRNA 失活率。使用内源性大肠杆菌 AAA + 蛋白酶特异的肽标签来诱导和调节 eGFP 的蛋白质降解率。测量Luc和eGFP的信使RNA失活率、蛋白质降解率、成熟时间。通过易于实施的定量程序，可以在无细胞反应中加速和调整全局 mRNA 周转率和蛋白质降解率，并将其调整到生物学相关范围内。这些功能拓宽了无细胞系统的能力，可以更好地控制基因表达。这种无细胞提取物可以在新的研究领域找到一些应用，例如体外合成生物学和系统生物学，其中工程信息过程需要对 mRNA 失活和蛋白质降解进行定量控制。
A large amount of recombinant proteins can be synthesized in a few hours with Escherichia coli cell-free expression systems based on bacteriophage transcription. These cytoplasmic extracts are used in many applications that require large-scale protein production such as proteomics and high throughput techniques. In recent years, cell-free systems have also been used to engineer complex informational processes. These works, however, have been limited by the current available cell-free systems, which are not well adapted to these types of studies. In particular, no method has been proposed to increase the mRNA inactivation rate and the protein degradation rate in cell-free reactions. The construction of in vitro informational processes with interesting dynamics requires a balance between mRNA and protein synthesis (the source), and mRNA inactivation and protein degradation (the sink). Two quantitative studies are presented to characterize and to increase the global mRNA inactivation rate, and to accelerate the degradation of the synthesized proteins in an E. coli cell-free expression system driven by the endogenous RNA polymerase and sigma factor 70. The E. coli mRNA interferase MazF was used to increase and to adjust the mRNA inactivation rate of the Firefly luciferase (Luc) and of the enhanced green fluorescent protein (eGFP). Peptide tags specific to the endogenous E. coli AAA + proteases were used to induce and to adjust the protein degradation rate of eGFP. Messenger RNA inactivation rate, protein degradation rate, maturation time of Luc and eGFP were measured. The global mRNA turnover and the protein degradation rate can be accelerated and tuned in a biologically relevant range in a cell-free reaction with quantitative procedures easy to implement. These features broaden the capabilities of cell-free systems with a better control of gene expression. This cell-free extract could find some applications in new research areas such as in vitro synthetic biology and systems biology where engineering informational processes requires a quantitative control of mRNA inactivation and protein degradation.