Elimination of Ribosome Inactivating Factors Improves the Efficiency of Bacillus subtilis and Saccharomyces cerevisiae Cell-Free Translation Systems
Elimination of Ribosome Inactivating Factors Improves the Efficiency of Bacillus subtilis and Saccharomyces cerevisiae Cell-Free Translation Systems
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DOI：
10.3389/fmicb.2018.03041
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发表时间：
2018-12-18
影响因子：
5.2
通讯作者：
Murina, Victoriia
中科院分区：
文献类型：
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作者：
Brodiazhenko, Tetiana;Johansson, Marcus J. O.;Murina, Victoriia
基于体外蛋白质合成优化的细胞裂解物的无细胞翻译系统在基础和应用科学中都有多种应用，从翻译调控的研究到蛋白质和核糖核酸上升链复合体的无细胞生产。为了在翻译系统中获得高活性和重复性，细胞裂解物中的核糖体具有酶活性是必不可少的。在这里，我们证明了编码核糖体失活因子的基因-枯草芽孢杆菌的HPF和酿酒酵母的Stmt-的基因组破坏有力地改善了细菌和酵母翻译系统的活性。重要的是，枯草杆菌HPF的消除会导致100S核糖体的完全丧失，否则会干扰基于二体的方法来制备用于冷冻电子显微镜研究的停滞不前的核糖体复合体。
Cell-free translation systems based on cellular lysates optimized for in vitro protein synthesis have multiple applications both in basic and applied science, ranging from studies of translational regulation to cell-free production of proteins and ribosomenascent chain complexes. In order to achieve both high activity and reproducibility in a translation system, it is essential that the ribosomes in the cellular lysate are enzymatically active. Here we demonstrate that genomic disruption of genes encoding ribosome inactivating factors - HPF in Bacillus subtilis and Stmt in Saccharomyces cerevisiae - robustly improve the activities of bacterial and yeast translation systems. Importantly, the elimination of B. subtilis HPF results in a complete loss of 100S ribosomes, which otherwise interfere with disome-based approaches for preparation of stalled ribosomal complexes for cryo-electron microscopy studies.