The cell free protein synthesis system from the model filamentous fungus Neurospora crassa

•A method to make a cell free protein synthesis (CFPS) system from Neurospora crassa.•Methods to analyze programmed mRNA translation in the N. crassa CFPS.•Application of CFPS to examine a translation regulation mechanism. 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.