Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70.
Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70.
复制标题
DOI：
10.1186/1754-1611-4-8
复制
发表时间：
2010-06-24
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
大肠杆菌无细胞表达系统利用噬菌体RNA聚合酶，如T7，合成大量的重组蛋白。这些系统在生物技术中被用于许多应用，例如蛋白质组学。最近，用无细胞系统在体外重建了信息过程。然而，这些合成方法由于缺乏转录模块化而受到严重限制。目前可用的无细胞系统已经针对噬菌体RNA聚合酶进行了优化，这对与生物信息相关的工程过程施加了很大的限制。为了在体外研究复杂的信息过程，需要开发具有更广泛转录能力的高效无细胞系统。在这项工作中，我们建立了一个高效的无细胞表达系统，该系统只使用内源性的大肠杆菌RNA聚合酶和Sigma因子70进行转录。分批生产了约0.75 mg/ml的萤火虫荧光素酶和增强绿色荧光蛋白。对含有不同调控部分的表达载体进行了优化，以提高表达水平。此外，还设计了一种新的绿色荧光蛋白，在无细胞系统中比原来的绿色荧光蛋白更具可译性。三种不同的三磷酸腺苷(ATP)再生系统：磷酸肌酸(CP)、磷酸烯醇式丙酮酸(PEP)和3-磷酸甘油酸(3-PGA)对蛋白质产量进行了表征。3-PGA的蛋白质产量最高。粗提物的制备被简化为一个简单的常规程序，包括细胞培养在内需要12个小时。尽管它使用内源性的大肠杆菌转录机制，但这个无细胞系统可以产生与噬菌体系统相当的活性蛋白质。大肠杆菌转录为在体外设计信息过程提供了更多的可能性。许多针对西格玛因子70的大肠杆菌启动子/操纵子是可用的，这些启动子/操纵子形成了一个广泛的调控部分文库。在这项工作中，无细胞表达被开发为一个工具箱，用于设计和研究体外合成的基因电路。
Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro. In this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP) regeneration systems: creatine phosphate (CP), phosphoenolpyruvate (PEP), and 3-phosphoglyceric acid (3-PGA). The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture. Although it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma factor 70 are available that form a broad library of regulatory parts. In this work, cell-free expression is developed as a toolbox to design and to study synthetic gene circuits in vitro.