state-of-the-art in vitro protein synthesis by

"Up to 100's of micrograms/ml in 240 minutes"

PUREfrex® Reconsituted, Recombinant Cell Free Protein Synthesis: System 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.

A few things you can do with PUREfrex.

• Produce prokaryotic proteins
• Produce eukaryotic proteins
• Produce membrane proteins
• Produce cytotoxic proteins.
• Produce disulfide-bonded proteins
• Confirm open reading frames
• Evaluate the effect of mutations on ORFs
• Evaluate effects of amino acid substitutions on protein activity
• Produce labeled proteins
• Add functional groups to proteins via unnatural amino acids.
• Produce truncated proteins for functional domain or epitope mapping
• Ribosome display
• Translation mechanisms research
• Synthetic biology
• Protein folding research
• In vitro compartmentalization

Ultra-high purification of PUREfrex components for minimal nuclease and protease activity

PUREfrex kit components are ultra-highly purified to remove contaminating nuclease and protease. Rigorous removal of nuclease and protease activity acts to preserve the integrity of DNA (or RNA) templates and to preserve the functionality of PUREfrex reaction proteins, resulting in maximal yield of fully intact target protein. Furthermore, with PUREfrex 2.0 and 2.1 kits, contamination of RNase and ß-galactosidase have been greatly reduced and lipopolysaccharide (LPS) has also been reduced to ~0.1 EU per 1 µL of reaction mixture.

PUREfrex kits are formatted for easy operation. One-step protocol amenable to high-throughput operations.

Base Purefrex kits contain only 4 vials. Solution I contains all non-protein components, Solution II contains all protein components, Solution III contains ribosomes. The fourth vial contains a ready-to-use DNA template for performing positive control reactions. To produce protein with Purefrex, simply mix your template with Solutions I, II and III and incubate.

Suitable templates can be prepared easily by PCR. Major requirements include a T7 promotor, a ribosome binding site (Shine-Delgarno sequence) and a stop codon. 

 

 

 

For detailed information and tips on template preparation, please click the link in the navigation sidebar on this page. 

PUREfrex protein components are not tagged.

As originally described, all PURE system protein components were HIS-tagged as an aid to purification of the target protein. Thus, after removal of ribosmes by centrifugation or filtration, a target protein could be quickly purified from PURE reaction components by negative selection, by passing the reaction mix over a metal ion affinity chromatography column. In the PUREfrex system, protein components are not tagged, leaving no questions about the effect of tagged translatin components on protein synthesis AND, enabling the user to produce target protein with any desired tag, or no tag at all, through template design.  

PUREfrex is based on PURE technology.

PUREfrex was introduced by Gene Frontier in 2011 as a commercial adaptation of the pioneering PURE system technology (protein using recombinant elements) developed by Professor Takuya Ueda at the University of Tokyo (ref 1, 2). PURE technology consists of factors for transcription, translation, aminoacylation, and energy regeneration. All protein factors and ribosomes are assembled with substrates including amino acids, NTPs, and tRNAs in buffer (see figure below). 

Translation factors include initiation factors IF1/IF2/IF3, elongation factors EF-Tu/EF-Ts/EF-G, release factors RF1/RF2/RF3, ribosome recycling factor (RRF), 20 aminoacyl-tRNA synthetases (ARS), and methionyl-tRNA transformylase. Thus PURE system has no unknown proteins and its composition can be adjusted freely. Further, the defined and modular chemistry of PURE and PUREfrex® systems offers extreme flexibility of the contents in the final reaction mix. 

Gene Frontier scientists have endeavored continuously to improve not only the protein synthesis capacity of PUREfrex® reactions, but also to expand the utility, applications and convenience of PUREfrex® kits. 

PURE System references
Shimizu et al. (2001) Nat. Biotechnol., vol. 19, p. 751. PubMed ID: 11479568
Shimizu et al. (2005) Methods, vol. 36, p. 299. PubMe ID: 16076456

Which PUREfrex Kit Is Right For Me?

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

To learn more about each version, use the links in the sidebar to go to a specific page. For pricing and ordering information, click the links in the PUREfrex Ordering Information table at the bottom of this page.

• 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 Version Comparison
PUREfrex Version	1.0	2.0/2.1
Specification
Yield of synthesized DHFR	150 ug/ml	600 ug/ml
Reaction volume required for 100ug DHFR	660 ul (> 3 kits)	160 ul (< 1 kit)
Yield of synthesized GFP	100 ug/ml	800 ug/ml
Reaction volume required for 100ug GFP	1000 ul (> 4 kits)	125 ul (< 1 kit)
Version Selection Guidance
Protein Preparation	--	++
Protein Science (translation, protein folding, etc.)	++	--
Synthetic Biology	++	+

PUREfrex® SUPPLEMENTS expand the potential of base Kits.

PUREfrex supplements add functionalities to PUREfrex reaction mixtures. Adding the right supplement could make the difference between success and failure to obtain acceptable yields of your target protein. Supplements are prepared in buffers and at concentrations that allow their use directly in standard PUREfrex reaction protocols. Like all PUREfrex protein components, all protein supplements are proteins are untagged. 

Molecular Chaperone Supplements to Promote Protein Solubility and Protein Folding
Molecular chaperones such as hsp70 (e.g. E. coli DnaK) assist the correct folding of the newly synthesized proteins. Because base PUREfrex® kits do not contain molecular chaperones, some proteins may have difficulty forming the correct conformation and be insoluble after synthesis. In such cases, molecular chaperones could contribute to make them soluble and active.

PUREfrex® Supplement: 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.

PUREfrex® Supplement: GroE Mix
GroE Mix comprises highly purified and ratio-optimized E. coli GroEL (known as Hsp60) and GroES (works in conjunction with GroEL). 

Redox Environment Supplements to Promote Disulfide Bond Formation
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.

PUREfrex® Supplement: 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.

PUREfrex® Supplement: PDI Set
PDI Set comprises oxidized glutathione (GSSG), human PDI (protein disulfide isomerase) and human Ero1α (ER oxidoreductin-1 to reoxidize PDI).

Elongation Factor P (EF-P) promotes translation of consecutive proline residues.
The E. coli translation factor EF-P (elongation factor P), a bacterial homolog of eukaryotic initiation factor 5A (eIF5A), functions to promote peptide bond formation between the first methionine and the next amino acid as well as the elongation of consecutive proline residues (3 or more residues). In bacteria, LYS34 of EF-P is modified post translationally to ß-lysllysine by EpmA (YjeA/GenX) and EpmB (YjeK). This modification is important to EF-P activity. PUREfrex EF-P Supplement contains this modification at LYS34.

PUREfrex® Supplement: EF-P
EF-P Supplement comprises recombinant EF-P (modified at LYS34) and dilution buffer. Standard PUREfrex® kits do not contain EF-P. Addition of EF-P to PUREfrex reactions can improve the yield of proteins containing consecutive proline residues such as Pro-Pro-Pro and Pro-Pro-Gly by preventing ribosome stalling and promoting the formation of peptide bonds.

---

PUREfrex Workflow 

A general scheme for using PUREfrex® to synthesize functional proteins is shown in the following figure.

Prepare a suitable template and perform a standard reaction. If insufficient protein is obtained, re-examine and re-design the template sequence. If needed, add supplements or modify reaction conditions. In general, one kit contains sufficient reagents to determine if the target protein can be synthesized.

---

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.