PURExpress is a novel coupled cell-free transcription/translation system reconstituted from purified components necessary for E.coli translation . Recombinant histidine-tagged aminoacyl-tRNA synthetases (20), initiation factors (3), elongation factors (3), release factors (3), ribosome recycling factor, methionyl-tRNA transformylase, T7 RNA polymerase, creatine kinase, myokinase, nucleoside-di-phosphate kinase, and pyrophophatase provide the activities required for coupled transcription and translation, as well as energy regeneration . Purified 70S ribosomes, amino acids, rNTP’s, and tRNA’s com-plete the system . PURExpress is based on the PURE system technology originally devel-oped by Dr . Takuya Ueda at the University of Tokyo and commercialized as the PURESYSTEM® by the Post Genome Institute (PGI) (Toyko, Japan) . PURExpress improves upon the PURESYSTEM® Classic II kit by optimiz-ing the components to increase the yield of protein synthesis . PURExpress is an easy-to-use one-step reaction that requires the mixing of only two tubes . Protein synthesis is initiated by the addition of template DNA and is largely complete within one hour . Products of translation can be analyzed by SDS-PAGE (Coomassie stained, autoradiograph of 35S-labeled proteins, or western blot) or in direct activity assays . Purification of the target protein can often be accomplished by ultrafiltration to remove the high MW ribosomes followed by IMAC (immobilized metal affinity chromatography) to remove the His-tagged components .Due to its reconstitution of recombinant components, PURExpress is es-sentially free of contaminating exonucleases, RNases, and proteases . Template DNA is not exposed to digestion and target proteins are free of post-transla-tional modifications (glycosylation, phosphorylation, and proteolysis) .