Skip to content


You currently have no items in your basket.

Total (excl. vat) £0.000
View basket & checkout

PUREfrex 2.1

The purest of the PURE-based reconstituted protein synthesis system kits with redox environment control


PUREfrex® is a reconstituted coupled transcription/translation system based on the PURE system originally developed by the Ueda group (Shimizu et al., 2001) and later commercialized as PURESYSTEM® (Shimizu and Ueda, 2010) by BioComber Co. Untagged protein components permit addition of any desired tag (including His-tags) to a protein of interest. For template generation we recommend Protein Express Linear Template PCR kits (available in two versions: His-tag only or ProX-tag + His-tag) or expression plasmids from Protein Express (pROX-FL92.1: ProX-tag + His-tag) or BioComber (pURE1-4: no tags).




All necessary macromolecular components needed for in vitro transcription and translation are highly purified from E. coli and supplied as two solution mixes (A and B). The system includes modules for aminoacylation, energy regeneration, transcription and translation.

The advantages of PUREfrex® include reduced levels of contaminating proteases, nucleases, and phosphatases, beta-galactosidase and LPS. Compared to cell lysate-based systems, the more defined and modular chemistry of PUREfrex® leads to greater yield, reproducibility and flexibility. PUREfrex® is available in two forms: PUREfrex® 2.0 and PUREfrex® 2.1. PUREfrex® 2.1 is identical to PUREfrex® 2.0 with the added ability for the user to control redox conditions; this is useful for proteins that require special redox conditions for proper folding and disulfide bond formation.

PUREfrex® 2.0 and PUREfrex® 2.1 contain no molecular chaperones. When proper folding, activity and solubility of your protein of interest requires molecular chaperones, Gene Frontier offers two molecular chaperone supplements for use with PUREfrex® 2.0 and PUREfrex® 2.1: GroE Mix and DnaK Mix. GroE Mix comprises an optimized mixture of highly purified E. coli GroEL (Hsp60) and GroES. DnaK Mix comprises an optimized mixture of highly purified E. coli DnaK, DnaJ and GrpE. DnaK (Hsp70) has ATPase activity and is stimulated by co-chaperones DnaJ and GrpE. DnaJ facilitates the ATPase activity of DnaK and can bind to hydrophobic protein regions. GrpE stimulates ADP/ATP exchange of DnaK. For a given protein the relative suitability of GroE Mix and DnaK Mix for promoting folding, solubility and activity must be determined empirically.

Gene Frontier also offers two additional supplements to promote proper disulfide bond formation: DS Supplement (soon to be phased out and replaced with DsbC Set) and PDI Set. Formation of disulfide bonds is important for the folding, stability and activity of many proteins, including cell surface receptors and secreted proteins. Disulfide bonds are formed by oxidation of pairs of cysteine sulfhydryl (SH-) groups. Therefore, an oxidizing environment is necessary for disulfide bond formation. Additionally, the correct pairing of cysteines is promoted by Disulfide bond isomerase (e.g. E. coli DsbC) which can catalyse disulfide bond exchange reactions. DS supplement comprises highly purified E. coli DsbC and oxidized Glutathione (CSSG). PDI Set comprises oxidised glutathione (GSSG), human PDI (Protein disulfide isomerase) and human Ero1α (ER oxidoreductin-1 to reoxidize PDI).

Benefits of PUREfrex®

  • PUREfrex® supplements expand the potential to express properly folded, disulfide-bonded, functional proteins.Molecular chaperones - Assists folding and improves solubility of proteins that require molecular chaperones for proper folding.
  • DnaK Mix – DnaK Mix comprises highly purified and ratio-optimized E.coli DnaK, DnaJ and GrpE. DnaK, known as Hsp70, has ATPase activity and is stimulated by co-chaperones DnaJ and GrpE. DnaJ facilitates DnaK ATPase activity and binds to hydrophobic regions of synthesized proteins. GrpE stimulates ADP/ATP exchange of DnaK. DnaK Mix works well with PUREfrex® series and DsbC Set.
  • GroE Mix - GroE Mix comprises highly purified and ratio-optimized E. coli GroEL (known as Hsp60) and GroES (working in conjunction with GroEL). GroE Mix works well with PUREfrex® series.
  • Disulfide bond enhancers - Formation of disulfide bonds is important for folding and stability of secretory proteins such as enzymes or antibodies. Disulfide bonds are usually formed by oxidation of sulfhydryl groups (SH-) of adjacent cysteine residues. Therefore, disulfide bond formation efficiency depends on redox state. Additionally, disulfide bond isomerase which can catalyze the exchange of disulfide bridges may be required for correct cysteine pairing.
  • DsbC Set - DsbC Set comprises highly purified E. coli DsbC (a disulfide bond isomerase) which can catalyze disulfide bridge exchange, and GSSG (oxidized Glutathione) to enforce an oxidized environment.
  • PDI Set - PDI Set comprises oxidized glutathione (GSSG), human PDI (protein disulfide isomerase) and human Ero1α (ER oxidoreductin-1 to reoxidize PDI).

Rapid synthesis of:

  • Membrane proteins
  • Antibodies
  • Antigens
  • Cytokines
  • Metal cofactor-dependent enzymes
  • Phosphoproteins
  • Antimicrobial peptides
  • Therapeutic peptides
  • All proteinaceous components of PUREfrex® are tag-free, permitting users to choose any desired tag for purification and monitoring.
  • Extremely low contamination from LPS, proteases, nucleases, β-galactosidase and phosphatases
  • Free of metabolic side reactions (found in cell extracts) that deplete amino acid pools
  • Flexibility of a modular system
  • Greater reproducibility resulting from more defined chemistry
  • Cleaner resolution of expressed products due to elimination of background proteins found in traditional cell extracts
  • Permits manipulation of protein synthesis reaction conditions, reactants, and direct monitoring of performance
  • Rapid production of ‘difficult’ proteins that may be toxic to conventional cell protein factories
  • Permits protein synthesis from different templates (DNA: circular plasmids, linear amplicons, rolling circle amplification; RNA: mRNAs)
  • High-throughput expression of multi-protein complexes

Generation of freeze-dryable point of use nanoscale biosensors:

  • Biosensors for endocrine disruptors
  • Biosensors for viruses
  • Biosensors for small molecules
  • Biosensors for quorum sensing molecules
  • Biosensor for histamin
  • Tetramerization-driven biosensor for anti-tag antibodies
  • High throughput functional genomics/proteomics
  • mRNA display
  • Ribosome display
  • Site-specific protein labelling/mutagenesis with non-natural amino acids. Cellular systems are susceptible to toxic side-effects of non-natural amino acids in cellular proteins or recombinant products; cell-free systems can be easily tailored to accommodate unusual side chain structures by modifying components of the translation machinery; for example, mutation of EF-Tu has been shown to improve the incorporation efficiency of non-natural amino acids with bulky side chains (Doi et al. 2007):

Biotin labelling

  • PEGylation
  • Post translational modification
  • Fluorescence labelling for molecular imaging
  • Double fluorescent labelling for single molecule FRET
  • Protein structure/function analysis
  • Stable isotopic labelling of internal standards for targeted quantitative proteomics (when proteins are expressed in living cells or cell extracts, labelling is complicated by metabolic processes that dilute isotope or scramble labelling patterns as a result of molecular transformations)
  • In vitro protein evolution
  • Rapid drug candidate screening
  • Rapid gene construct screening
  • Rapid confirmation of coding potential of ORFs
  • Study of artificial cells
  • Study of protein translation
  • Study of protein folding
  • Study of protein engineering
  • Study of ribosome function
  • Study of ribosome stalling

Synthesis of E.coli phosphatase (AppA)
E. coli phosphatase (AppA) was synthesized with PUREfrex® 2.1 and DS supplement at 37°C for 4 hours. Protein yield was similar under different redox states, but protein activity was highest with 4 mM GSH and 2 mM GSSG (oxidized glutathione).


Synthesis of IgG
Immunoglobulin G (IgG) is a Y-shaped tetramer comprising two heavy chains (HC) and two light chains (LC) joined by disulfide bonds. HCs and LCs were synthesized in a single tube with PUREfrex® 2.1, DS supplement and DnaK Mix at 37°C for 16 hours. Although the yields of HCs and LCs were similar across all conditions, the yield of tetrameric IgG was highly dependent on the nature and amount of reducing agent.