1. About PUREfrex®
PUREfrex® is a reconstituted cell-free protein#PF005-0.5-EX
For 500 µL Reaction
PUREfrex® is NOT included.

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

GSSG (Green)*1

May 2023

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

25µL

Store at -80 ºC before opening

25µL

E.coli DsbC (320 μM)*3Store at -80ºC

Dilution Buffer (Clear) 500 µL
30% glycerol bufferStore at -20ºC*1)Standard final concentration of GSSG is 3 mM.
We recommend the optimizing the concentration of any reducing agent to achieve higher activity because it is depends on thesynthesized protein.
*2)
The concentration unit has been changed from “mg / mL” to“µM” . 7.5mg / mL is equal to 320µM.
Standard final concentration of DsbC is 1 - 16 μM.
We recommend the optimizing the concentration of any reducing agent to achieve higher activity because it is depends on thesynthesized protein.
Please use attached Dilution Buffer for dilution of DsbC.
*3)
For storage at -80ºC, the rest of solution should be frozen rapidly
in liquid nitrogen or dry ice/ethanol. Divide into aliquots, if necessary, and avoid refreeze and thaw as much as possible.

DsbC Set is developed for in vitro research use only.
DsbC 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 DsbC 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

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

Store at -20ºC*2Formation 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.

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

Oxidized glutathione (60 mM)

DsbC (Green)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.

2. About DsbC Set / PDI Set(Materials)
PUREfrex® 2.1 (#PF213) contains Solution I, Solution II,Solution III, Cysteins and GSH.
DsbC Set (#PF005) contains DsbC and GSSG.
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 DsbC on ice.
3. Mix each solution by vortex and centrifuge briefly tocollect each solution at the bottom.
4. Dilute DsbC 4-fold with Dilution Buffer.
5. Assemble the reaction mixture in a tube as follows.
(Add the template DNA to 1-3 ng/µL per 1 kbp)

Phone: +81-3-5632-9610

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

FAX: +81-3-5632-9619Water
Solution I *4
10 mM Cysteine
80 mM GSH
60 mM GSSG
Solution II
Solution III
80 µM DsbC *5
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

This is a standard protocol for synthesizing proteins
containing disulfide bonds. Each solution of DsbC Set andPUREfrex® 2.1 (#PF213) are mixed together in a sametube. For example, 20 µL of reaction is assembled asbelow, which final concentration of each reagent will be
0.5 mM Cysteine, 4 mM GSH and 3 mM GSSG and 4 μM
DsbC.

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