|PF005-0.5-EX||DS supplement||500 µL for reaction||–||
“DS supplement” is the supplement for synthesizing proteins containing a disulfide bond in an active form with PUREfrex®.
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.
DS supplement is constituted of highly purified DsbC from E.coli known as disulfide bond isomerase, which can catalyze the disulfide bridge exchange, and GSSG (oxidized Glutathione) to make an oxidized environment.
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) and DS supplement (#PF005) enables an aggregate-prone protein to be synthesized in a soluble form.
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.
|GSSG||25 μL||Oxidized glutathione (60 mM)||-20 °C|
|DsbC||25 μL||E.coli DsbC (7.5 mg/mL) (No tags)||-80 °C *2|
|Dilution buffer||500 μL||30% glycerol buffer||-20 °C|
This is a standard protocol for synthesizing proteins containing disulfide bonds.
Each solution of DS supplement (#PF005) and PUREfrex® 2.1 (#PF213) are mixed together in a same tube.
- 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 each 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 *1 8 μL 10 mM Cysteine 1 μL 80 mM GSH 1 μL 60 mM GSSG 1 μL Solution II 1 μL Solution III 2 μL DsbC (1.875 mg/mL) *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 the synthesized products. Please add the same amount of H2O to the reaction for the sample of SDS-PAGE.
DS supplement is developed for in vitro research only. DS supplement 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-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”