PUREfrex Product Pages

PUREfrex Product Manual and Package Inserts

Key Technical Notes

PUREfrex FAQ

PUREfrex in the Literature

Manufacturer's Site Links

Safety Data Sheets

Posters

  • General Protein Synthesis
    • Improvement of translational efficiency by N-terminal codon optimization in the reconstituted cell-free protein synthesis system [2016]
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    • Improvement of the translation efficiency of proline residue-containing proteins using the reconstituted cell-free protein synthesis system (PUREfrex) [2018] VIEW
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    • Investigation on how to synthesize active proteins by using a reconstituted cell-free protein synthesis system (PUREfrex) [2020]
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  • Disulfide Bonding
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    • Synthesis of proteins containing disulfide bonds using a reconstituted cell-free protein synthesis system (PUREfrex). [2019]
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    • Synthesis of proteins containing disulfide bonds using PUREfrex® with human protein disulfide isomerase [2018] VIEW
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    • Synthesis of functionally active proteins containing disulfide bonds using the new PURE system (PUREfrex) [2011]
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  • Synthesis of Immunoglobulins and Biologics
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    • Unique Cell-Free Protein Synthesis System, PUREfrex -Useful Platform for Protein Expression in the Development of Biologics- [2018] VIEW
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    • Preparation of labeled cyclic peptides with PUREfrex [2017]
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    • Development of a method for the synthesis of aglycosylated full-length IgG using PUREfrex [2017]
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    • Efficient in vitro expression of aglycosylated full-length IgG using a reconstituted cell-free protein synthesis system, PUREfrex 2.0 [2016]
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    • In vitro production of Fab, Toxin, and Immunotoxin using PUREfrex®2.0 with increased productivity [2015]
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    • Synthesis of functionally active proteins containing disulfide bonds using the new PURE system (PUREfrex) [2011]
Purefrex®Dashboard

PUREfrex® 2.1

Reconsituted, Recombinant Cell Free Protein Synthesis

Overview

PUREfrex® is an ultra-cool, ultra-smart, and ultra-useful in vitro coupled transcription/translation system based on E.coli translation machinery but completely different from S30 translation extracts. PUREfrex® is NOT AN EXTRACT. PUREfrex® is a RECONSTITUTED SYSTEM comprising highly purified E. coli ribosomes, highly purified recombinant proteins, and other highly purified factors required for transcription, translation, aminoacylation, and energy regeneration. As such, PUREfrex kits provide previously unthinkable reaction controllability compared to cell extract-based systems and a well defined mix of molecular components from which to purify synthesized protein.

PUREfrex®2.1 Ordering Information
Product NameCatalog NumberTotal Reaction Volume
PUREfrex® 2.1 GFK-PF213-0.25-EX 250 ul
GFK-PF213-2ML-EX 1 x 2 ml
GFK-PF213-10ML-EX 5 x 2 ml
 GFK-PF213-50ML-EX  25 x 2 ml
Standard kit for protein synthesis when disulfide bonding is desired. Redox agent (DTT) omitted from Solution 1. Redox agents are individually vialed.   

Which PUREfrex Kit Is Right For Me?

PUREfrex® coupled transcription/translation system kits are available in four versions.

PUREfrex® 2.0 mini -PUREfrex 2.0 mini is an entry model designed for those who are trying protein synthesis for the first time with a cell-free system.
PUREfrex® 2.0 - Choose 2.0 when protein yield is you primary goal and disulfide bonding is not relevant.
PUREfrex® 2.1 - 2.1 kits are based on 2.0 reaction components but are not premixed with reducing agent. 2.1 kits include separate vials of cysteine, DTT, and reduced glutathione allowing control of the redox environment. Choose 2.1 kits for primarily for target proteins with disulfide bonds.
PUREfrex® 1.0 - 1.0 kits produce less protein than 2.X kits, but the complete contents of 1.0 kits is disclosed. Chose 1.0 when complete information on PUREfrex reaction components takes precedence over protein yield.
PUREfrex® 1.0 Custom - Gene Fronteir is happy to provide custom reaction mixtures whenever feasible. Custom PUREfrex kits are based on 1.0 reaction components. Please contact us with your request.

Please Note: All protein components in all PUREfrex versions are tag free allowing the synthesis of protein with any desired tag.

About PUREfrex® 2.1

PUREfrex®2.1 is newly launched in 2017 to make it possible for selecting a different reducing agent, while keeping other composition of PUREfrex®2.0.

PUREfrex® 2.1 vs PUREfrex® 2.0

PUREfrex®1.0 was launched in 2011. An upgraded version PUREfrex®2.0 with improved performance was launched in 2015.

PUREfrex®2.1 was launced in 2017. As implied by the version number, 2.1 is based on 2.0 components but is formatted differently to allow convenient user control of the reducing agent to aid in finding experimental conditions that best promote the desired disulfide bonding configuration of the target protein.

Formation of a disulfide bond is an important process for folding and stability of secretory proteins such as enzymes or antibodies. A disulfide bond is usually formed by the oxidation of sulfhydryl group (SH-) of adjacent cysteine residues. Therefore, the formation efficiency of a disulfide bond depends on redox state. Additionally, disulfide bond isomerase which can catalyze the exchange of disulfide bridges may be also required for a correct pairing of cysteines.

The redox state in the reaction mixture of PUREfrex® is affected by the type of reducing agent and the ratio of reducing and oxidizing agent. PUREfrex®2.0 contains dithiothreitol (DTT) in Solution I as reducing agent. In contrast, it is possible to select a suitable reducing agent for the protein of your interest by using PUREfrex®2.1, in which reducing agent is not added to Solution I. PUREfrex®2.1 includes Solution I neither DTT nor Cysteine. Each solution of cysteine, DTT and reduced glutathione (GSH) is attached to the kit independently. Other reducing agents such as 2-mercaptoethanol can be also used.

PUREfrex 2.1 Kit Contents and Storage(Catalog Number: GFK-PF213-0.25-EX)
ReagentQuantityDescriptionStorage *1
Solution Ⅰ 100 μL Amino acids, NTPs, tRNAs and substrates for enzymes etc. -20 °C
Solution Ⅱ 12.5 µL Proteins in 30 % glycerol buffer -20 °C or -80 °C *2
Solution Ⅲ 25 µL Ribosome (20 µM) -80 °C *2
Cysteine 20 μL Cysteine (10 mM) -20 °C
DTT 20 μL Dithiothreitol (40 mM) -20 °C
GSH 20 μL Reduced glutathione (80 mM) -20 °C
DHFR DNA *3 10 µL PCR product containing a gene encoding dihydrofolate reductase (DHFR) from E.coli as a positive control. (20 ng/μL) -20 °C
  • ＊1) Indicated temperature shows storage temperature after opening the kit. Please store the kit at -80 °C before opening.
  • ＊2) The rest of solution should be frozen rapidly in liquid nitrogen or dry ice/ethanol, and be stored at -80 °C. Divide into aliquots, if necessary, and avoid refreeze and thaw as much as possible.
  • ＊3) For synthesizing DHFR, add 1 µL of DHFR DNA to 20 µL of reaction. Sequence of DHFR DNA is here.
Purefrex Kit Contents: 2.0 vs. 2.1
2.0 kits contain Cysteine and DTT in Solution 1. These are omitted in 2.0 Solution 1. 
Reagent Name Description v2.0 v2.1
Solution
1		 Amino acids, NTPs, tRNAs, enzyme substrates, etc. 125 ul 100 ul
Solution
II		 Proteins (dissolved in 30% glycerol solution) 12.5 ul 12.5 ul
Solution
III		 20 uM Ribosomes 25 ul 25 ul
Cysteine 10 mm Cysteine not incl. 20 ul
DTT 40 uM Dithiothreitol not incl 20 ul
GSH
80 mM Reduced Glutathione
 not incl. 20 ul
DHFR DNA Template DNA containing gene encoding E. coli dihydrofolate reductase (DHFR)
for positive control of protein synthesis (20 ng/uL)   		 10 ul 10 ul

PUREfrex 2.1 Reaction Protocol For Proteins Containing Disulfide Bonds

The following is a standard protocol for synthesizing proteins containing disulfide bonds. Therefore DsbC Set is also used with PUREfrex® 2.1. GSSG and DsbC are components of the DsbC Set product. For synthesis of proteins without disulfide bonds, please add DTT instead of GSH and do not use DsbC Set components.

  1. 1) Thaw Solution I, Cysteine, GSH and GSSG by incubation at room temperature or 37 °C for 1 minute for completely dissolving, and then cool on ice.
  2. 2) Thaw Solution II, III and DsbC on ice.
  3. 3) Mix each solution by vortex and centrifuge briefly to collect solution at the bottom.
  4. 4) Assemble the reaction mixture in a tube as follows. Add the template DNA to 1 - 3 ng/µL per 1 kb
Contents of a standard 20 ul PUREfrex 2.1 reaction for proteins containing disulfide bonds volume
Water 5-X µL
Solution I 8 µL
10 mM Cysteine 1 µL
80 mM GSH 1 µL
60 mM GSSH (oxidized glutatihione) 1 µL
Solution II 1 µL
Solution III 2 µL
80 uM DsbC (dilute with buffer from DsbC Set) 1 µL
Template DNA * X µL
Total 20 µL
* Please visit the template DNA preparation site

PUREfrex Ordering Information

PUREfrex Ordering Information
Product Name
(click for price and order info)		Catalog NumberTotal Reaction VolumeNotes
Entry Model Kits
PUREfrex® 2.0 mini GFK-PF201-0.1-EX 100 μL New item !
PUREfrex® 2.0 mini is an entry model designed for those who are trying protein synthesis for the first time with a cell-free system.Product Information Page：PUREfrex® 2.0 mini
Kits
PUREfrex® 2.0 GFK-PF201-0.25-EX 250 ul  
GFK-PF201-0.25-5-EX 5 x 250 ul (packaged as 5 x 250 ul) Sales to be discontinued
GFK-PF201-2ML-EX 1 x 2 ml  
GFK-PF201-10ML-EX 5 x 2 ml Note: cat. no. change from GFK-PF201-10-EX
GFK-PF201-50ML-EX 25 x 2 ml New size !
Current generation kit for protein synthesis. Contains DTT as a reducing agent. Product Information Page：PUREfrex® 2.0
PUREfrex® 2.1 GFK-PF213-0.25-EX 250 ul  
GFK-PF213-0.25-5-EX 5 x 250 ul (packaged as 5 x 250 ul) Sales to be discontinued
GFK-PF213-2ML-EX 1 x 2 ml New size!
GFK-PF213-10ML-EX 5 x 2 ml Note: cat. no. change from GFK-PF213-10-EX
GFK-PF213-50ML-EX 25 x 2 ml New size !
Current generation kit for proteins with disulfide bonds. Formulated for user control of redox environment. Product Information Page：PUREfrex® 2.1
PUREfrex® 1.0 GFK-PF001-0.25-EX 250 ul  
GFK-PF001-0.25-5-EX 1250 ul (packaged as 5 x 250 ul)  Sales to be discontinued
GFK-PF001-2ML-EX 1 x 2 ml  New size!
 GFK-PF001-10ML-EX 5 x 2 ml Note: cat. no. change from GFK-PF001-10-EX
 GFK-PF001-50ML-EX 25 x 2 ml New size !
Prior generation kit for protein science research and synthetic biology. Components are fully disclosed. Product Information Page：PUREfrex® 1.0
Custom formulated kits are based on 1.0 components. Please inquire about PUREfrex® custom.
PUREfrex Reaction Supplements
DnaK Mix GFK-PF003-0.5-EX for 500 ul of reaction mix  
GFK-PF003-10ML-EX 5 x 2 ml  Note: cat. no. change from GFK-PF003-10-EX
GFK-PF003-50ML-EX 25 x 2 ml New size !
GroE Mix GFK-PF004-0.5-EX for 500 ul of reaction mix  
PF004-10ML-EX 5 x 2 ml  Note: cat. no. change from GFK-PF004-10-EX
PF004-50ML-EX 25 x 2 ml New size !
Supplements for synthesis of aggregate-prone proteins. Product Information Page：PUREFrex Reaction Supplements
DsbC Set GFK-PF005-0.5-EX for 500 ul of reaction mix  
PF005-10ML-EX 5 x 2 ml Note: cat. no. change from GFK-PF005-10-EX
PF005-50ML-EX 25 x 2 ml New size !
PDI Set GFK-PF006-0.5-EX for 500 ul of reaction mix   
PF006-10ML-EX 5 x 2 ml Note: cat. no. change from GFK-PF006-10-EX
PF006-50ML-EX 25 x 2 ml New size !
Supplements for synthesis of proteins containing disufide bonds.. Product Information Page：PUREFrex Reaction Supplements
EF-P GFK-PFS052-0.5-EX for 500 ul of reaction mix   
PFS052-10ML-EX 5 x 2 ml Note: cat. no. change from GFK-PFS052-10-EX
PFS052-50ML-EX 25 x 2 ml New size !
Supplement for use when consecutive prolines are in target protein.  Product Information Page：PUREFrex Reaction Supplements

Notes

PUREfrex® is developed for in vitro research only. PUREfrex® should not be used for the therapy, diagnostic or administration to animals including human and should not be used as food or cosmetics etc. To avoid the contamination of nuclease, nuclease-free-treated water, reagents and materials should be used. We also recommend wearing gloves and mask.
“PUREfrex® is Registered in U.S. Patent and Trademark Office”