About PUREfrex®

#PF213-10ML-EX

For 2 mL x 5 Reaction

in vitro research use only
Store at -80ºC before opening
GeneFrontier Corporation
www.genefrontier.com

May 2023

SHARP Kashiwa Building, 4F
273-1 Kashiwa, Kashiwa-shi,
Chiba 277-0005 Japan

Solution I *1

800 µL x 5

Amino acids, NTPs, tRNAs and substrates of enzymes
Store at -20ºC

Solution II *2

100 µL x 5

Proteins in 30% glycerol bufferStore at -20ºC or -80ºC *3

Solution III *2

200 µL x 5

Ribosomes (20 µM)
Store at -80ºC *3

Cysteine *1

160 µL x 5Cysteine (10 mM)
Store at -20ºC

DTT *1160 µL x 5

Dithiothreitol (40 mM)
Store at -20ºC

GSH *1160 µL x 5

Reduced glutathione (80 mM)
Store at -20ºC

DHFR DNA*2, 4

10 µL x 1

PCR product (20 ng/µL) containing a gene encoding E. coli
DHFRStore at -20ºC

PUREfrex® is a reconstituted cell-free protein
synthesis kit which GeneFrontier has developed based on
the PURE system technology. The target protein can besynthesized by adding the template DNA (or mRNA) tothe reaction mixture. The PURE system is a unique cell-freeprotein synthesis system, which has originally developedby Professor Takuya Ueda at the University of Tokyo, andc o n s i s t s o f o n l y p u r i fi e d f a c t o r s n e c e s s a r y f o rtranscription, translation and energy regeneration (Ref. 1).
Therefore it enables to adjust the composition of thereaction mixture.
PUREfrex® has been raised in purity by improving themethods for preparing ribosomes, tRNAs and all proteinsin the reaction mixture compared with the original PURE
system (Ref. 2). As the result, the contaminating
lipopolysaccharide from E. coli is reduced to around 0.1 EU
per 1 µL of reaction and other contaminants, such as
RNase and β-galactosidase, are also reduced.
Because all of proteins in PUREfrex® have no tags, thesynthesized protein can be purified and detected by anytags.

1. Construction of the template DNA
T7 promoter

Gene of Interest

STOPRBS(Ribosome Binding Site; SD sequence)Store at -80 ºC before opening*2)
Solution II, Solution III and DHFR DNA are the same as PUREfrex® 2.0 (#PF201).
*3)
For storage at -80ºC, the remaining solution should be frozen
rapidly in liquid nitrogen or dry ice/ethanol. Please divide intoaliquots, if necessary, and avoid refreeze and thaw as much aspossible.
*4)
As a positive control for the protein synthesis reaction, 1 µL ofDHFR DNA should be added to 20 µL of reaction. Please visit ourweb site to get the nucleic acid sequence of DHFR DNA.

An example of the method for preparing the template
DNA by two-step PCR is shown in below.
FOR primer
REV primer
T7PRO-SD primer

PCRThe template DNA for the protein synthesis byREV primerPUREfrex® should contain T7 promoter and ribosome
binding site (SD sequence) at the upstream of the geneencoding the target protein.
At the downstream of stop codon, more than 10
nucleotides are required.
All three stop codons are available.
Both circular and linear DNAs are available as a templateDNA.
For the circular DNA, e.g. plasmid DNA, T7 terminator isrequired at the downstream of stop codon.
For the linear DNA, e.g. PCR product or DNA fragment
digested by restriction enzymes, T7 terminator is notnecessarily required.

T7 promoterRBSPCR
Gene of Interest

3. Sequence of primers
FOR primer
5’-AAGGAGATATACCA-ATG-N(10-20)-3’RBS

REV primer
5’-GGATTAGTTATTCA-TTA-N(10-20)-3’
more than 10 any nucleotides

T7PRO-SD primer
5’-GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCC(References)
1. Shimizu et al. (2001) Nat. Biotechnol., vol. 19, p. 751.
2. Shimizu et al. (2005) Methods, vol. 36, p. 299.

*1)
Reducing agents are excluded from Solution I in PUREfrex® 2.1.
Please add attached Cysteine and reducing agent (DTT or GSH) tothe reaction mixture.
The standard working concentration of Cysteine, DTT and GSH
are 0.5, 2 and 4 mM, respectively. Other reducing agents are alsoavailable for the reaction, e.g. 2-mercaptoethanol and TCEP.
We recommend optimizing the concentration of reducing agent
to synthesize a protein with higher activity because the suitableconcentration is depends on the synthesized protein.

2. Prepatation of the template DNA

T7 promoter
TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCA-3’RBS

The protein synthesis reaction using PUREfrex® 2.1
can be performed in any volume. For example, 20 µL ofreaction mixture containing 0.5 mM Cysteine and 4 mM
GSH is assembled as below.
1. Thaw completely Solution I, Cysteine and GSH byincubation at room temperature or 37 ºC for 1 minute,and then cool on ice.
2. Thaw Solution II and Solution III on ice.
3. Mix each solution by vortex and centrifuge briefly tocollect solution at the bottom.
4. Assemble the reaction mixture in a tube as follows.
(Add the template DNA to 0.5-3 ng/µL per 1 kbp)
Water
7 -X µL
Solution I8 µL*5)
Cysteine (10 mM)
1 µL
GSH (80 mM)
1 µL
Solution II
1 µL
Solution III
2 µL
Template DNA
X µL
Total
20 µL
5. Incubate the tube at 37ºC for 2-6 hours.
6. Analyze the synthesized product.
*5)
Please note that the volume of Solution I in PUREfrex® 2.1(#PF213) is different from PUREfrex® 2.0 (#PF201).

PUREfrex® is developed for in vitro research use only.
PUREfrex ® s h o u l d n o t b e u s e d f o r t h e t h e r a p y ,diagnostic or administration to animals including humanand should not be used as food or cosmetics etc.
To avoid the contamination of nuclease, nuclease-freetreated water, reagents and materials should be used. We
also recommend wearing gloves and mask.
PUREfrex ® is a registered trademark of GeneFrontier
Corporation. For any commercial use of PUREfrex ® ,please contact GeneFrontier in advance.
e-mail : purefrex@genefrontier.com【Outside Japan】【JAPAN】

Carlsbad, CA 92010, USA

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email: info@cosmobiousa.com

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Phone: +81-3-5632-9610
FAX: +81-3-5632-9619

URL: https://www.cosmobio.co.jp/