The E. coli S30 lysate proteome: A prototype for cell-free protein production
The E. coli S30 lysate proteome: A prototype for cell-free protein production
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DOI：
10.1016/j.nbt.2017.09.005
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发表时间：
2018-01-25
影响因子：
5.4
通讯作者：
Bernhard, Frank
中科院分区：
文献类型：
--
作者：
Foshag, Daniel;Henrich, Erik;Bernhard, Frank
使用加工的细胞裂解物生产蛋白质是合成生物学的核心技术，这些系统非常适合生产困难的毒素或膜蛋白。然而，无细胞系统的中央裂解液的组成仍然是一个“黑匣子”。大肠杆菌裂解物的无细胞表达效率最高，每毫升反应可产生数毫克蛋白质。它们的制备意味着蛋白质组分级分离，从而产生强烈偏差且未知的裂解物成分。许多代谢途径预计将被截断或完全消除。缺乏对基本无细胞裂解物蛋白质组的了解是定向裂解物工程方法以及使用未纯化反应混合物进行测定设计的主要瓶颈。这项研究通过提供源自常用大肠杆菌菌株 A19 的 S30 裂解物蛋白质组的蓝图，开始弥补这一差距。 S30 裂解物经常用于无细胞蛋白质生产，是大多数商业大肠杆菌无细胞表达系统的基础。 821 种蛋白质的一部分被鉴定为 S30 裂解物中的核心蛋白质组，约占已知大肠杆菌蛋白质组的四分之一。它将其分类为与转录/翻译、折叠、稳定性和代谢过程相关的功能组，将为定制的无细胞反应构建框架。作为一个例子，我们展示了培养过程中 SOS 反应诱导导致调整后的 S30 裂解物具有更好的折叠能力，并提高了合成蛋白质的溶解度和活性。所提供的数据和协议可以作为生成定制无细胞系统和产品分析的平台。
Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a "black box". Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures.This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as a platform for the generation of customized cell-free systems and product analysis.