Mechanistic Insights into Cell-Free Gene Expression through an Integrated -Omics Analysis of Extract Processing Methods

Cell-free systems derived from crude cell extracts have
developed
into tools for gene expression, with applications in prototyping,
biosensing, and protein production. Key to the development of these
systems is optimization of cell extract preparation methods. However,
the applied nature of these optimizations often limits investigation
into the complex nature of the extracts themselves, which contain
thousands of proteins and reaction networks with hundreds of metabolites.
Here, we sought to uncover the black box of proteins and metabolites
in Escherichia coli cell-free reactions
based on different extract preparation methods. We assess changes
in transcription and translation activity from σ70 promoters in extracts prepared with acetate or glutamate buffer
and the common post-lysis processing steps of a runoff incubation
and dialysis. We then utilize proteomic and metabolomic analyses to
uncover potential mechanisms behind these changes in gene expression,
highlighting the impact of cold shock-like proteins and the role of
buffer composition.

以粗制细胞提取物为基础构建的无细胞系统，现已发展成为基因表达研究的成熟工具，可应用于原型开发、生物传感与蛋白质生产领域。此类系统研发的核心在于细胞提取物制备方法的优化。然而，这类优化的应用导向属性，往往限制了对提取物本身复杂特性的深入研究——这类提取物包含数千种蛋白质，以及涵盖数百种代谢物的复杂反应网络。本研究旨在揭开基于不同提取物制备方法的大肠杆菌（Escherichia coli）无细胞反应体系中蛋白质与代谢物组成的“黑箱”。我们评估了以乙酸盐或谷氨酸盐缓冲液制备，且经过洗脱孵育、透析这两种常见裂解后处理步骤的提取物中，源自σ70启动子的转录与翻译活性变化。随后，我们借助蛋白质组学与代谢组学分析手段，解析此类基因表达活性变化背后的潜在调控机制，阐明了冷激样蛋白的关键作用以及缓冲液组成的具体影响。