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The cell free protein synthesis system from the model filamentous fungus Neurospora crassa
Methods ( IF 4.8 ) Pub Date : 2018-03-01 , DOI: 10.1016/j.ymeth.2017.12.003
Cheng Wu 1 , Ananya Dasgupta 1 , Lunda Shen 1 , Deborah Bell-Pedersen 1 , Matthew S Sachs 1
Affiliation  

Cell-free protein synthesis (CFPS) can be used in many applications to produce polypeptides and to analyze mechanisms of mRNA translation. Here we describe how to make and use a CPFS system from the model filamentous fungus Neurospora crassa. The extensive genetic resources available in this system provide capacities to exploit robust CFPS for understanding translational control. Included are procedures for the growth and harvesting of cells, the preparation of cell-free extracts that serve as the source of the translational machinery in the CFPS and the preparation of synthetic mRNA to program the CFPS. Methods to accomplish cell-free translation and analyze protein synthesis, and to map positions of ribosomes on mRNAs by toeprinting, are described.

中文翻译:

来自模型丝状真菌粗糙脉孢菌的无细胞蛋白质合成系统

无细胞蛋白质合成 (CFPS) 可用于许多应用,以生产多肽和分析 mRNA 翻译机制。在这里,我们描述了如何从模型丝状真菌粗糙脉孢菌中制作和使用 CPFS 系统。该系统中可用的广泛遗传资源提供了利用强大的 CFPS 来理解转化控制的能力。包括细胞的生长和收获程序、作为 CFPS 中翻译机制来源的无细胞提取物的制备以及用于对 CFPS 进行编程的合成 mRNA 的制备。描述了完成无细胞翻译和分析蛋白质合成的方法,以及通过 toeprinting 在 mRNA 上绘制核糖体位置的方法。
更新日期:2018-03-01
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