Brodiazhenko Tetiana, Johansson Marcus J O, Takada Hiraku, Nissan Tracy, Hauryliuk Vasili, Murina Victoriia
Department of Molecular Biology, Umeå University, Umeå, Sweden.
Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.
Front Microbiol. 2018 Dec 18;9:3041. doi: 10.3389/fmicb.2018.03041. eCollection 2018.
Cell-free translation systems based on cellular lysates optimized for protein synthesis have multiple applications both in basic and applied science, ranging from studies of translational regulation to cell-free production of proteins and ribosome-nascent 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 and Stm1 in - robustly improve the activities of bacterial and yeast translation systems. Importantly, the elimination of 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.
基于为蛋白质合成优化的细胞裂解物的无细胞翻译系统在基础科学和应用科学中都有多种应用，范围从翻译调控研究到蛋白质和核糖体 - 新生链复合物的无细胞生产。为了在翻译系统中实现高活性和可重复性，细胞裂解物中的核糖体具有酶活性至关重要。在这里，我们证明编码核糖体失活因子的基因（酿酒酵母中的HPF和裂殖酵母中的Stm1）的基因组破坏显著提高了细菌和酵母翻译系统的活性。重要的是，消除HPF会导致100S核糖体完全丧失，否则这些核糖体会干扰用于冷冻电子显微镜研究的停滞核糖体复合物制备的双体方法。
