Description
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 complete the system. PURExpress is based on the PURE system technology originally developed 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 optimizing 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 essentially free of contaminating exonucleases, RNases, and proteases. Template DNA is not exposed to digestion and target proteins are free of post-translational modifications (glycosylation, phosphorylation, and proteolysis).