PURESYSTEM® is a novel reconstituted in vitro translation system consisting of about 30 purified enzymes necessary for transcription, translation and energy recycling. All factors for transcription and translation are 6X His-tagged: 3 initiation factors (IF1, IF2, IF3), 3 elongation factors (EF-G, EF-Tu, EF-Ts), 3 release factors (RF1, RF2, RF3), Ribosome recycling factor, 20 aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase and T7 RNA polymerase. Also included in non-tagged form are E. coli 70S ribosomes, amino acids, NTPs, E. coli tRNA and an energy recycling system. For template generation we recommend a Protein Express Linear Template PCR kit (use the no His-tag option of the His-tag version or expression plasmids from BioComber (pURE1-4: no tags).
Depicted is the use of PURESYSTEM® to synthesize and purify untagged target proteins from linear PCR-based templates or expression plasmids. We recommend expression plasmids from BioComber (pURE1-4).
Example of expression and reverse purification of DHFR produced in PURESYSTEM®
Examples of fluorescent lysine-labeled proteins produced in PURESYSTEM®. Proteins were separated on a 10-20% gradient SDS PAGE gel, stained with SYPRO® Red and analyzed on a fluorescent gel scanner. Red bands: PURESYSTEM® component proteins. Green bands: Synthesized target proteins.