|PF213-0.25-EX||PUREfrex®2.1||250 µL for reaction||–||
|PF213-0.25-5-EX||PUREfrex®2.1||1250 µL for reaction||–||
PUREfrex® was launched in 2011, then we improved the protein productivity of PUREfrex® and the upgraded version “PUREfrex® 2.0” (#PF201) has been available since 2015.
PUREfrex® 2.1 is newly launched in 2017 to make it possible for selecting a different reducing agent, while keeping other composition of PUREfrex® 2.0.
Formation of a disulfide bond is an important process for folding and stability of secretory proteins such as enzymes or antibodies. A disulfide bond is usually formed by the oxidation of sulfhydryl group (SH-) of adjacent cysteine residues. Therefore, the formation efficiency of a disulfide bond depends on redox state. Additionally, disulfide bond isomerase which can catalyze the exchange of disulfide bridges may be also required for a correct pairing of cysteines.
The redox state in the reaction mixture of PUREfrex® is affected by the type of reducing agent and the ratio of reducing and oxidizing agent. PUREfrex® 2.0 contains dithiothreitol (DTT) in Solution I as reducing agent. In contrast, it is possible to select a suitable reducing agent for the protein of your interest by using PUREfrex® 2.1, in which reducing agent is not added to Solution I. PUREfrex® 2.1 includes Solution I neither DTT nor Cysteine. Each solution of cysteine, DTT and reduced glutathione (GSH) is attached to the kit independently. Other reducing agents such as 2-mercaptoethanol can be also used.
DS supplement (#PF005-0.5-EX)
This supplement includes Oxidized glutathione (GSSG) and DsbC protein (E. coli disulfide bond isomerase).
Using DS supplement with PUREfrex® 2.1 (#PF213) enables a protein containing disulfide bonds to be synthesized in an active form.
DnaK Mix (#PF003-0.5-EX)
This supplement includes the mixture of DnaK, DnaJ and GrpE, which are molecular chaperone and cofactors in E. coli.
Using DnaK Mix with PUREfrex® 2.1 (#PF213) enables an aggregate-prone protein to be synthesized in a soluble form.
This supplement works very well when it is used with DS supplement (#PF005).
Applications showed in below indicate that the selection of the suitable reducing agent and its ratio with oxidizing agent is very important to synthesize a protein containing disulfide bonds with an active form.
Synthesis of E.coli acid phosphatase (AppA)
AppA contains four consecutive and one nonconsecutive disulfide bonds. Its productivity and enzyme activity were compared when it was synthesized using PUREfrex® 2.1 with DTT or GSH as reducing agent.
AppA was synthesized with PUREfrex® 2.1 and DS supplement at 37 °C for 4 hours. As a result, the productivity was almost the same under different redox state, but the product synthesized with 4 mM GSH and 2 mM GSSG showed the highest activity.
Synthesis of IgG
Active immunoglobulin G (IgG) forms a “Y”-shaped conformation, which consists of two heavy chains (HC) and two light chains (LC) connected by disulfide bonds. The productivity of HC and LC and the formation efficiency of Y-shaped IgG were compared when they were synthesized using PUREfrex® 2.1 with different reducing agents.
HC and LC were synthesized in the single tube with PUREfrex® 2.1, DS supplement and DnaK Mix at 37 °C for 16 hours. As a result, although the synthesis amounts of HC and LC were almost the same, the formation efficiency of IgG was different depending on the reducing agent.
Please visit our web site to download the poster titled “Development of a method for the synthesis of aglycosylated full-length IgG using PUREfrex ” for more details.
|Solution I||100 μL||Amino acids, NTPs, tRNAs and substrates for enzymes etc.||-20 °C|
|Solution II||12.5 μL||Proteins in 30 % glycerol buffer||-20 °C or -80 °C *2|
|Solution III||25 μL||Ribosome (20 μM)||-80 °C *2|
|Cysteine||20 μL||Cysteine (10 mM)||-20 °C|
|DTT||20 μL||Dithiothreitol (40 mM)||-20 °C|
|GSH||20 μL||Reduced glutathione (80 mM)||-20 °C|
|10 μL||PCR product containing a gene encoding dihydrofolate reductase (DHFR) from E.coli as a positive control. (20ng/μL)||-20 °C|
This is a standard protocol for synthesizing proteins containing disulfide bonds. Therefore DS supplement (#PF005) is also used with PUREfrex® 2.1. GSSG and DsbC are included in DS supplement. When you synthesize proteins without disulfide bonds, please add DTT instead of GSH and no DS supplement.
- Thaw Solution I, Cysteine, GSH and GSSG by incubation at room temperature or 37 °C for 1 minute completely and then cool on ice.
- Thaw Solution II, III and DsbC on ice.
- Mix each solution by vortex and centrifuge briefly to collect solution at the bottom.
- Assemble the reaction mixture in a tube as follows. Add the template DNA to 1 – 3 ng/µL per 1 kb.
Water 5-X μL Solution I 8 μL*1 10 mM Cysteine 1 μL 80 mM GSH 1 μL 60 mM GSSG 1 μL Solution II 1 μL Solution III 2 μL 1.875 mg/mL DsbC *2 1 μL Template DNA*3 X μL Total 20 μL
- Incubate the tube at 37 °C for 2 – 6 hours with heat block or water bath.
Protein synthesis reaction is almost done until 6 hours, but some proteins require longer incubation (e.g. 24 hours) to form disulfide bonds between the correct pair of cysteine residues.
- Analyze or use the synthesized products.
PUREfrex® is developed for in vitro research only. PUREfrex® should not be used for 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-free-treated water, reagents and materials should be used. We also recommend wearing gloves and mask.
“PUREfrex® is Registered in U.S. Patent and Trademark Office”