#PF201-0.25-EX

For 250 µL Reaction

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

May 2023

1. Overview

2-2. Preparation of the template DNAPUREfrex® is a reconstituted cell-free protein synthesis

An example of preparation of the template DNA by PCR
is shown below. Nucleic acid sequences of the primers areshown in 2-3. The elements shown in 2-1 are necessary
for the template DNA, even using the other preparing
methods.

kit which GeneFrontier has developed based on the PURE
system technology. The PURE system is a cell-free protein
synthesis system, which has originally developed byProfessor Takuya Ueda at the University of Tokyo, and isc o n s i s t e d 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).
The target protein is synthesized by adding the template
DNA (or mRNA) to the reaction mixture. PUREfrex ® isconsisted of only purified factors, therefore it enables toadjust the composition of the reaction mixture.
PUREfrex ® is raised in the purity by improving thepreparation methods of 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 below 0.1 EU per
1 µL of reaction and other contaminants, such as RNase
and β-galactosidase, are also reduced.
In the PUREfrex ® , all proteins have no tags for
purification or detection, therefore the target protein
would be synthesized and purified by any tag.

3. Mix Solution I, II and III by vortex and centrifuge briefly
to collect each solution at the bottom.

PCR

REV primer

T7PRO-SD primer

REV primer

PCRReferences) 1. Shimizu et al. (2001) Nat. Biotecnol., vol. 19, p. 751

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

1. Thaw Solution I by incubation at 30ºC for 1 minute,and then cool on ice.
2. Thaw Solution II and III on ice.

FOR primer

T7 promoter

The protein synthesis reaction using PUREfrex ® isscalable. For example, 20 µL of reaction is assembled asbelow.

Gene of Interest

4. Assemble the reaction mixture in a tube as follows.
(Add the template DNA to 0.5-3 ng/µL per 1 kbp)
Water
Solution I
Solution II
Solution III
Template DNATotal

7 -X µL
10 µL
1 µL
2 µL
X µL
20 µL

5. Incubate the tube at 37ºC for 2-4 hours.
6. Analyze the synthesized product.

RBS

2. Shimizu et al. (2005) Methods, vol. 36, p. 299

Store at -80ºC before opening

Solution I (White)

2-1. Construct of the template DNA

125 µL

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

Solution II (Black)

Store at -20ºC or -80ºC

Solution III (Red)*125 µL

Ribosomes (20 µM)
Store at -80ºC*2*110 µL

PCR product (20 ng/µL) containing a gene encoding E. coli
DHFRStore at -20ºC*1)
For storage at -80ºC, the rest of solution should be frozen rapidly inliquid nitrogen or dry ice/ethanol. Divide into aliquots, if necessary,and avoid refreeze and thaw as much as possible.
*2)
As a positive control for the protein synthesis reaction, 1.0 µL ofDHFR DNA should be added to 20 µL of reaction. Please visit ourweb site to get the nucleic acid sequence of DHFR DNA.

FOR primer

Gene of Interest
ATG

12.5 µL

Proteins in 30% glycerol buffer

DHFR DNA (Clear)

T7 promoter

2-3. Sequences of primers

STOPRibosome Binding Site(SD sequence)
The template DNA for the protein synthesis byPUREfrex® should contain T7 promoter and ribosome
binding site (SD sequence) at the upstream of the geneencoding the target protein. All three stop codons areavailable. More than 10 nucleotides are needed after thestop codon.
Both circular and linear DNAs are available as a templateDNA. For the circular DNA, T7 terminator is required at
the downstream of the stop codon. For the linear DNA,
which includes a PCR product and a DNA digested by a
restriction enzyme, T7 terminator is not necessarily
required at the 3’ -terminus.

5’-AAGGAGATATACCA-ATG-N(10-20)-3’RBS

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

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

T7PRO-SD primer
5’-GAAATTAATACGACTCACTATAGGGAGACC
T7 promoter

ACAACGGTTTCCCTCTAGAAATAATTTTGTTTA
ACTTTAAGAAGGAGATATACCA-3’RBS【Outside Japan】【JAPAN】

Carlsbad, CA 92010, USA

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email: info@cosmobiousa.com

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