Garenne David, Beisel Chase L, Noireaux Vincent
School of Physics and Astronomy, University of Minnesota, 115 Union Street SE, Minneapolis, MN, 55455, USA.
Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Center for Infection (HZI) Research, 97080, Würzburg, Germany.
Rapid Commun Mass Spectrom. 2019 May 15;33(11):1036-1048. doi: 10.1002/rcm.8438.
Cell-free transcription-translation (TXTL) is becoming a popular technology to prototype and engineer biological systems outside living organisms. TXTL relies commonly on a cytoplasmic extract that provides the molecular components necessary to recapitulate gene expression in vitro, where most of the available systems are derived from E. coli. The proteinic and enzymatic composition of lysates, however, is typically unknown. In this work, we analyzed by mass spectrometry the molecular constituents of the all-E. coli TXTL platform myTXTL prepared from the E. coli strain BL21 Rosetta2.
Standard TXTL reactions were assembled and executed for 10-12 hours at 29°C. In addition to a no-DNA control, four DNA programs were executed in separate reactions to synthesize the reporter protein deGFP as well as the phages MS2, phix174 and T7. The reactions were treated according to standard procedures (trypsin treatment, cleaning) before performing liquid chromatography/mass spectrometry (LC/MS). Data analysis was performed using Sequest and protein identification using Scaffold.
A total of 500-800 proteins were identified by LC/MS in the blank reactions. We organized the most abundant protein sets into several categories pertaining, in particular, to transcription, translation and ATP regeneration. The synthesis of deGFP was easily measured. The major structural proteins that compose the three phages MS2, phix174 and T7 were also identified.
Mass spectrometry is a practical tool to characterize biochemical solutions as complex as a cell-free TXTL reaction and to determine the presence of synthesized proteins. The data presented demonstrate that the composition of TXTL based on lysates can be used to validate some underlying molecular mechanisms implicated in cell-free protein synthesis. The composition of the lysate shows significant differences with respect to similar studies on other E. coli strains.
无细胞转录-翻译（TXTL）正成为一种在活生物体之外对生物系统进行原型设计和工程改造的流行技术。TXTL通常依赖于一种细胞质提取物，该提取物提供了在体外重现基因表达所需的分子成分，目前大多数可用系统都源自大肠杆菌。然而，裂解物的蛋白质和酶组成通常是未知的。在这项工作中，我们通过质谱分析了由大肠杆菌BL21 Rosetta2菌株制备的全大肠杆菌TXTL平台myTXTL的分子成分。
组装标准的TXTL反应，并在29°C下进行10 - 12小时。除了无DNA对照外，在单独的反应中执行四个DNA程序，以合成报告蛋白deGFP以及噬菌体MS2、phix174和T7。在进行液相色谱/质谱（LC/MS）分析之前，按照标准程序（胰蛋白酶处理、清洗）对反应进行处理。使用Sequest进行数据分析，并使用Scaffold进行蛋白质鉴定。
通过LC/MS在空白反应中总共鉴定出500 - 800种蛋白质。我们将最丰富的蛋白质组分为几类，特别是与转录、翻译和ATP再生相关的类别。deGFP的合成很容易测量。还鉴定出了构成三种噬菌体MS2、phix174和T7的主要结构蛋白。
质谱是一种实用工具，可用于表征像无细胞TXTL反应这样复杂的生化溶液，并确定合成蛋白质的存在。所呈现的数据表明，基于裂解物的TXTL组成可用于验证无细胞蛋白质合成中涉及的一些潜在分子机制。裂解物的组成与对其他大肠杆菌菌株的类似研究相比存在显著差异。
