1. About PUREfrex®
PUREfrex® is a reconstituted cell-free protein synthesis#PF006-12ML-EX
For 2 mL x 6 Reaction
PUREfrex® is NOT included.

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

GSSG

*1

2024 Summer
SHARP Kashiwa Building, 4F
273-1 Kashiwa, Kashiwa-shi,
Chiba 277-0005 Japan

100 µL x 6

Oxidized glutathione (60 mM)
Store at lower than -20ºC

PDI

*2

100 µL x 6

Human protein disulfide isomerase (200 µM)*3Store at -80ºC

Ero1α

*2

100 µL x 6Human ER oxidoreductin-1 (5 µM)*3kit which GeneFrontier has developed based on the PURE
system technology. The target protein can be synthesized
by adding the template DNA (or mRNA) to the reaction
mixture. The PURE system is a unique cell-free protein
synthesis system, which has originally developed byProfessor 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.

500 µL x 1
Store at lower than -20ºC

Formation of a disulfide bond is one of an important
process for folding and stability of most of secretory
proteins such as enzymes or antibodies.
Oxidative environment is necessary to form a disulfide
bond because a disulfide bond is formed by the oxidationof sulfhydryl groups (-SH) of adjacent cysteine residues.
Disulfide bond isomerase, which can catalyze theexchange of disulfide bridges, may be also required for acorrect pairing of cysteines.
DsbC Set (#PF005) includes oxidized glutathione (GSSG)
and E. coli DsbC protein (disulfide bond isomerase in theperiplasm of E. coli).
PDI Set (#PF006) includes oxidized glutathione (GSSG),
human PDI (protein disulfide isomerase) and human Ero1α (ER oxidoreductin-1 to reoxidize PDI).
Addition of DsbC Set or PDI Set to PUREfrex® enables aprotein containing disulfide bonds to be synthesized in an
active form.
Efficiency of the formation of disulfide bonds isdependent on reducing agent in the reaction mixture. We
recommend the use of PUREfrex® 2.1 (#PF213) in which
the suitable reducing agent can be selected.

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

Store at -80 ºC before opening*1)Standard final concentration of GSSG is 1 – 3 mM with 4 mM GSH(reduced glutathione) in PUREfrex 2.1 (#PF213).
We recommend to check the optimal concentration of GSSG
because it depends on the target protein and the kind and concentration of reducing agent.
*2)Standard final concentration of PDI/Ero1α is 1/0.025 - 10/0.25 µM.
We recommend to check the optimal concentration of PDI andEro1α because it depends on the target protein.
Please use attached Dilution Buffer for diluting PDI and Ero1α.
*3)
For storage at -80ºC, the rest of solution should be frozen rapidly
in liquid nitrogen or dry ice/ethanol. Please divide into aliquots, if
necessary, and avoid refreeze and thaw as much as possible.

PDI Set is developed for in vitro research use only. PDI
Set 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-freetreated water, reagents and materials should be used. We
also recommend wearing gloves and mask.
For information concerning commercial use of PDI Set,
please contact GeneFrontier.
e-mail : purefrex@genefrontier.com【Outside Japan】【JAPAN】

Carlsbad, CA 92010, USA

KOTO-KU. TOKYO 135-0016, JAPAN2792 Loker Ave West, Suite 101
email: info@cosmobiousa.com

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

Store at -80ºC

Dilution Buffer

2. About DsbC Set / PDI Set

1. Thaw Solution I, Cysteine, GSH and GSSG by incubation
at room temperature or 37 ºC for 1 minute completely,and then cool on ice.
2. Thaw Solution II, III and PDI on ice.
3. Mix each solution by vortex and centrifuge briefly tocollect each solution at the bottom.

Phone: +81-3-5632-9610

Phone/FAX: (+1) 760-431-4600

FAX: +81-3-5632-9619

Water
Solution I *4
10 mM Cysteine
80 mM GSH
20 mM GSSG *5
Solution II
Solution III
PDI
Template DNATotal

5-X µL
8 µL
1 µL
1 µL
1 µL
1 µL
2 µL
1 µL
X µL
20 µL

URL: www.cosmobiousa.com

Here is a standard protocol for synthesizing proteins
containing disulfide bonds using PDI Set and PUREfrex®
2.1 (#PF213). For example, please assemble 20 µL ofr e a c t i o n m i x t u r e a s b e l o w , i n w h i c h t h e fi n a lconcentration of each reagent is 0.5 mM Cysteine, 4 mM
GSH, 1 mM GSSG and 10 µM PDI.
Ero1α can be also used instead of GSSG. Standard molar
concentration ratio of PDI:Ero1α is 40:1. For example, add
0.25 µM Ero1α with 10 µM PDI to the reaction mixture.

TOYO EKIMAE BLDG. 2-20, TOYO 2-CHOME,

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

6. Incubate the tube at 37ºC for 2-6 hours.
Protein synthesis reaction is almost done until 6 hours,
but some proteins require longer incubation (e.g. 24hours) to form disulfide bonds between the correct pairof cysteine residues.
7. Analyze the synthesized product.

4. Dilute GSSG 3-fold with water.
5. Assemble the reaction mixture in a tube as follows.
(Add the template DNA to 1-3 ng/µL per 1 kbp)

*4)
Please note that the volume of Solution I of PUREfrex® 2.1 (#PF213)
is different from PUREfrex® 2.0 (#PF201).
*5)
Please use 3-fold diluted GSSG.