PURESYSTEM®is anE. colireconstituted coupled transcription/translation system first commercialized in 2010 by BioComber (Tokyo, Japan). It consists of about 30 purified enzymes necessary for transcription, translation and energy recycling. All factors for transcription and translation are tagged withhexahistidine. These incude3 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. PURESYSTEM®also contains E. coli 70S ribosomes, amino acids, NTPs, E. coli tRNAs and an energy recycling system, so target proteins can be synthesized just by addition of template DNA (or mRNA). The hexahistidine tags permit rapid and simple reverse purification of synthesized proteins by immobilized metal affinity chromatography followed by untrafiltration to remove high MW ribosomes.The PURESYSTEM Delta (Component Dropout)series of products are PURESYSTEM®kits in which one or two components are excluded. The excludedcomponents are also available as individual supplements (Suppli series of products) for add back to cognate PURESYSTEM Delta series kits. Together, thePURESYSTEM Delta and Suppli series synergize to permit maximum flexibility, especially in studies of protein translation.PURESYSTEM® can be used to express proteins containing unnatural amino acids by exploitingProtein Express'sCloverDirect™line of tRNA reagents for site-directed protein functionalization.CloverDirect™ UAGor CGGGaminoacyl tRNAs are available charged with different unnatural amino acids that confer a range of functionality, including fluorescent-labeling, biotin-labeling,PEGylation, chemical crosslinking and photo-regulation. Targeting incorporationofspecific unnatural amino acids is achieved by designing proteinexpression templates with UAG and/or CGGG codons at user-defined positions.For template construction, we recommend Protein Express Linear Template PCR kits (no His-tag option of His-tag version) or BioComber Universal Primer; or, alteranatively,expression plasmids from BioComber (pURE1-4: no tags). The ProX tag is a specialNterminal peptide designed for the incorporation of certain large unnatural amino acids that require precise Nterminal positioning (codon 9) and a specific codon context (flanked by AAC and TCT).Codon 9 of the ProX tag can be varied betweenUAG, CGGG or TTT (as a control).PURESYSTEM®kits are only available as Delta (ComponentDropout) Kits.