Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA.
Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA.
Methods. 2018 Mar 15;137:11-19. doi: 10.1016/j.ymeth.2017.12.003. Epub 2017 Dec 30.
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.
无细胞蛋白质合成（CFPS）可用于多种应用，以生产多肽并分析 mRNA 翻译的机制。在这里，我们描述了如何从模式丝状真菌粗糙脉孢菌（Neurospora crassa）中构建和使用 CFPS 系统。该系统具有丰富的遗传资源，为理解翻译控制提供了强大的 CFPS 利用能力。其中包括细胞生长和收获的程序、用作 CFPS 中转录机制来源的无细胞提取物的制备以及用于编程 CFPS 的合成 mRNA 的制备。描述了进行无细胞翻译和分析蛋白质合成的方法，以及通过足迹印迹法在 mRNA 上定位核糖体位置的方法。
