A rapid method for gene expression analysis, PURExpress is a novel cell-free transcription/translation system reconstituted from the purified components necessary for E. coli translation. Transcription and translation are carried out in a one-step reaction, which requires the mixing of only two tubes. Protein synthesis is initiated by the addition of template DNA and is typically complete within two hours. 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 has minimal 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).

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Protein Expression Using the PURExpress® In Vitro Protein Synthesis Kit
25 µl reactions containing 250 ng template DNA were incubated at 37°C for 2 hours. 2.5 µl of each reaction was analyzed by SDS-PAGE using a 10–20% Tris-glycine gel. Note that proteins can be purified using reverse affinity chromatography (reagents not supplied). The red dot indicates the protein of interest. Marker M is the Protein Ladder (NEB #P7703)
PUREXPRESS® is a registered trademark of New England Biolabs, Inc..
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