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Cell-Free Protein Synthesis Enhancement from Real-Time NMR Metabolite Kinetics: Redirecting Energy Fluxes in Hybrid RRL Systems
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2017-10-02 00:00:00 , DOI: 10.1021/acssynbio.7b00280
Baptiste Panthu 1 , Théophile Ohlmann 2 , Johan Perrier 3 , Uwe Schlattner 4 , Pierre Jalinot 1 , Bénédicte Elena-Herrmann 3 , Gilles J. P. Rautureau 3
Affiliation  

A counterintuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity has an upper limit at native ribosome concentration and that lower amounts of the ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.

中文翻译:

实时NMR代谢动力学从无细胞蛋白质合成的增强:重定向混合RRL系统中的能量通量。

一种违反直觉的无细胞蛋白质合成(CFPS)策略,基于减少兔子网状细胞裂解液(RRL)中的核糖体组分,触发了由无RRL无核糖体上清液与来自不同哺乳动物细胞类型的核糖体互补组成的杂交系统的发展。混合RRL系统保持原始核糖体细胞类型的翻译特性,并提供类似于RRL的蛋白质表达水平。在这里,我们表明,消耗ATP的持久性核糖体相关代谢活动是最大蛋白质产量的主要障碍。我们提供基于代谢物动力学的实时核磁共振(NMR)研究的混合CFPS系统能量代谢的详细图片。我们证明蛋白质合成能力在天然核糖体浓度下具有上限,而核糖体级分的较低量优化了朝蛋白质翻译的能量通量,因此增加了CFPS的产量。这些结果为进一步的哺乳动物CFPS开发提供了合理化的策略,并揭示了实时NMR代谢表型对优化无细胞蛋白质表达系统的潜力。
更新日期:2017-10-02
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