The Escherichia coli-based reconstituted cell-free protein synthesis system, which we named the PURE (Protein synthesis Using Recombinant Elements) system, provides several advantages compared with the conventional cell-extract-based system. Stability of RNA or protein is highly improved because of the lack of harmful degradation enzymes. The system can be easily engineered according to purposes or the proteins to be synthesized, by manipulating the components in the system. In this chapter, we describe the construction and exploitation of the PURE system. Methods for preparing and assembling the components composing the PURE system for the protein synthesis reaction are shown.