state-of-the-art protein synthesis by

Overview
PUREfrex® is an in vitro coupled transcription/translation system based on E.coli translation machinery but completely different from S30 translation extracts. PUREfrex® is reconstituted from highly purified E. coli ribosomes, highly purified recombinant proteins, and other highly purified factors required for protein translation in vitro. PUREfrex® is an evolution of the PURE (protein from recombinant elements) system technology developed by Professor Takuya Ueda at the University of Tokyo. 

Purefrex 2.1 Ordering Information
Product Name	Product Number	Total Reaction Volume per Kit
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
Standard kit for protein synthesis when disulfide bonding is desired. Redox agent (DTT) omitted from Solution 1. Redox agents are individually vialed.

PUREfrex Kit Selection

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

• PUREfrex® 2.0 - use 2.0 when protein yield is the primary goal and disulfide bonding is not relevant.
• PUREfrex® 2.1 - use 2.1 when protein disulfide bonding is required.  
• PUREfrex® 1.0 - use 1.0 when complete information on PUREfrex reaction components is more important than protein yield. 
• PUREfrex® 1.0 Custom - inquire about custom kits to alter the components included in PUREfrex 1.0 reaction solutions.   

For more detailed information about PUREfrex®, click here

Which PUREfrex Kit Is Right For Me?

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

• 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.  

For detailed information about each PUREfrex® version, please use the navigation links in the sidebar on this page. 

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
Reagent	Quantity	Description	Storage ^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
Indicated temperature shows storage temperature after opening the kit. Please store the kit at -80 °C before opening. 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. For DHFR synthsis, use 1 µL of DHFR DNA in a 20 µL of reaction. DHFR DNA sequence.

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. 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. Thaw Solution II, III and DsbC on ice.
3. Mix each solution by vortex and centrifuge briefly to collect solution at the bottom.
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
Product Name (click for price and order info)	Catalog Number	Total Reaction Volume	Notes	Product Information Page
Kits
PUREfrex 2.0	GFK-PF201-0.25-EX	250 ul		2.0
	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	New size !
	GFK-PF201-10ML-EX	5 x 2 ml	Note: cat. no. change from GFK-PF201-10-EX
Current generation kit for protein synthesis. Contains DTT as a reducing agent.
PUREfrex 2.1	GFK-PF213-0.25-EX	250 ul		2.1
	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
Current generation kit for proteins with disulfide bonds. Formulated for user control of redox environment.
PUREfrex 1.0	GFK-PF001-0.25-EX	250 ul		1.0
	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
Prior generation kit for protein science research and synthetic biology. Components are fully disclosed. Custom formulated kits are based on 1.0 components. Please inquire about custom kits.
PUREfrex Reaction Supplements
DnaK Mix	GFK-PF003-0.5-EX	for 500 ul of reaction mix		Supplements
	GFK-PF003-10ML-EX	5 x 2 ml	Note: cat. no. change from GFK-PF003-10-EX
GroE Mix	GFK-PF004-0.5-EX	for 500 ul of reaction mix		Supplements
	PF004-10ML-EX	5 x 2 ml	Note: cat. no. change from GFK-PF004-10-EX
Supplements for synthesis of aggregate-prone proteins.
DsbC Set	GFK-PF005-0.5-EX	for 500 ul of reaction mix		Supplements
	PF005-10ML-EX	5 x 2 ml	Note: cat. no. change from GFK-PF005-10-EX
PDI Set	GFK-PF006-0.5-EX	for 500 ul of reaction mix		Supplements
	PF006-10ML-EX	5 x 2 ml	Note: cat. no. change from GFK-PF006-10-EX
Supplements for synthesis of proteins containing disufide bonds.
EF-P	GFK-PFS052-0.5-EX	for 500 ul of reaction mix		Supplements
	PFS052-10ML-EX	5 x 2 ml	Note: cat. no. change from GFK-PFS052-10-EX
Supplement for use when consecutive prolines are in target protein.

 PUREfrex® is 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 a registered trademark of the Gene Frontier Corporation. All rights reserved. 2021.