A newly developed reconstituted cell-free protein synthesis system

PUREfrex® 2.0


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Background

PUREfrex® kit is a newly developed reconstituted cell-free protein synthesis reagent based on PURE system technology invented by Professor Takuya Ueda in the University of Tokyo.

The reaction system consists of proteins, ribosome, amino acids and NTPs only (Ref. 1, 2). Those proteins are necessary for transcription, translation and energy regeneration. The proteins and ribosomes are highly purified individually and assembled together to constitute the protein synthesis system. To synthesize your protein, just add your template DNA or mRNA encoding the protein of interest into reaction mixture, and incubate for several hours. This system’s biggest point is RECONSTITUTED system by assembling translation related factors only. By this unique character, you may adjust the composition of reaction mixture as you like and may not have to consider serious background for your downstream application.

By improving the purification process of components of PUREfrex® kit, contamination of RNase and β-galactosidase are greatly reduced, in addition to that, lipopolysaccharide (LPS) is also reduced less than 0.1 EU per 1 µL of reaction mixture. All proteinous components of PUREfrex® kit have no tags for purification and detection. It allows to fuse your protein with any tag to purify the product.

 

There are 2 types of kit, so you may choose the kit depend on your protein’s properties!

PUREfrex® (#PF201-0.25-EX) has a reducing agent (DTT), so the protein is synthesized under reduced environment. We recommend PUREfrex® kit for synthesis of intracellular proteins or peptides which don’t form disulfide bonds (SS-bond).

PUREfrex® (#PF201-0.25-EX) with DS Suppliment is provided for synthesizing protein under oxidized environment. Oxidizing agent (GSSG) and disulfide bond isomerase (E.coli DsbC) are attached to PUREfrex® kit (#PF201-0.25-EX) as a supplement independently. We recommend PUREfrex® (#PF201-0.25-EX) with DS Suppliment for synthesis of an extracellular proteins or peptides which form disulfide bonds.

If you don’t know whether your protein needs disulfide bonds or not for its activity, you may synthesize it with different concentration of GSSG or DsbC using Solution IV and V attached to them. Please refer to our site showing the experiment using proteins including multiple disulfide bounds in active form.

Application

For preparation of following proteins
prokaryotic protein
eukaryotic protein
membrane protein
protein containing disulfide bonds
protein containing unnatural amino acids
... etc.
For basic research in protein science
Translation
Folding of protein after synthesis
For in vitro display
Ribosome display
mRNA display

Feature and Advantages

PUREfrex® kit is a reagent pursued of the original concept of PURE system technology, which is “reaction system consisting of translation system only”. So, one of the big improvements of PUREfrex® kit is improved purity of components of the kit. Table 1 shows the methods for preparing PUREfrex® kit compared with that for original PURE system. Especially, lipopolysaccharide (LPS) is reduced less than 0.1 EU per 1 µL of reaction mixture of PUREfrex®. Also, contamination of RNase and β-galactosidase are reduced (Fig. 1).

In addition to that, we reviewed the construct of each components for better activity, so all components of PUREfrex® kit have no tags for purification and detection. It allows fusion of your protein with any tag. The result of purification of the synthesized protein with histidine tag is shown in Fig. 2.

Table1

Fig1

To compare the amounts of contaminants in the reaction mixture of PUREfrex® and original PURE system, the amount of LPS and the activity of RNase and β-galactosidase were measured and compared as relative value. The amount and activity of original PURE system (Ref. 2) are set to 100. The amounts of all the contaminants of PUREfrex® are lower than those of original PURE system.

Fig2

GFP with histidine tag (His-tag) was synthesized using PUREfrex® and purified with metal-chelating resin.
Fig. 2 shows synthesized His-tagged protein can be purified with high purity by just one step.

PUREfrex® 2.0 and DS supplement

Version UP!! PUREfrex® 2.0 Series! Now is trial term!

Greatly improved synthsis quantity!
Smaller size and lower price trial products are available on trial term.


ESynthesis quantity is greatly improved. (- x10times)

EImproved the composition of the reaction reagent, keeping the PUREfrex features (Reducing contamination of RNase and β-galactosidase and lipopolysaccharide (LPS)), and improved efficiency of synthesis.

E Term of guarantee is 4months.

E Smaller size and lower price for easy trial.

E For 200ul reaction (Version 1 is for 500ul reaction)

 

 

 

Kit component

For 250 µL Reaction
ReagentQuantityDescriptionStorage
Solution I (White) 125 µL Amino acids, NTPs, tRNAs and substrates for enzymes etc. -20Ž
Solution II (Black) 12.5 µL Proteins in 30% glycerol buffer -20Ž or -80Ž–1
Solution III (Red) 12.5 µL x2 Ribosomes (20 µM) -80Ž–1
DHFR DNA (Clear)–2
Sequence itextj
10 µL PCR product (20 ng/µL) containing a gene encoding E.coli DHFR.

-20Ž
1) The rest of solution should be frozen rapidly in liquid nitrogen or dry ice/ethanol, and be stored at -80Ž. Divide into aliquots, if necessary, and avoid refreeze and thaw as much as possible.
2) For synthesizing DHFR, add 2.5 μL of DHFR DNA to 50 μL of reaction.

Others

References

1. Shimizu et al. (2001) Nat. Biotechnol., vol. 19, p. 751
2. Shimizu et al. (2005) Methods, vol. 36, p. 299

Product List

Product Name Cat# Quantity Price

PUREfrex(R)2.0 DataSheet ,

GFK-PF201-0.25-EX

1 KIT ¥15,000
$200

PUREfrex(R)2.0 DataSheet ,

GFK-PF201-0.25-5-EX

1 KIT ¥65,000
$867

DS supplement DataSheet ,

GFK-PF005-0.5-EX

1 KIT ¥6,000
$80

GroE Mix DataSheet ,

GFK-PF004-0.5-EX

1 KIT ¥11,000
$147

DnaK Mix DataSheet ,

GFK-PF003-0.5-EX

1 KIT ¥11,000
$147

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.



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