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PUREfrex®SS is a newly developed reconstituted cell-free protein synthesis reagent based on PURE system technology. PUREfrex®SS includes oxidized glutathione (GSSG; Solution IV) and DsbC protein (disulfide bond isomerase; Solution V) as a supplement with PUREfrex® (# PF001). PUREfrex®SS enables proteins including disulfide bonds (SS-bond) to be synthesized in active form.
For some proteins, such as secretory proteins to the extracellular, formation of disulfide bond is important for folding and stability. Disulfide bonds are usually formed from the oxidation of sulfhydryl (SH-) groups of adjacent cysteine residues. So, oxidized environment is necessary to form disulfide bonds. Additionally, disulfide bond isomerase which can catalyze the disulfide bridge exchange is also necessary to form a correct paring of cysteines. Using PUREfrex®SS, the protein can be synthesized under oxidized environment with isomerase.

*1) Indicated temperature shows storage temperature after opening the kit. Please store the kit at -80°C before opening.
*2) The rest of solution should be frozen rapidly in liquid nitrogen or dry ice/ethanol, and be stored at -80°C. Divide into aliquots, if necessary, and avoid refreeze and thaw as much as possible.
*3) For synthesizing DHFR, add 2.5 µL of DHFR DNA to 50 µL of reaction.

Protocol

The amounts given are for a 50 µL reaction. For scaling up the reaction, adjust the volume of reagents accordingly.
Thaw Solution I and IV by incubation at 30°C for 1minute, and then cool on ice.
Thaw Solution II, III and V on ice.
Mix Solution I, II, III, IV and V respectively 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 1kb.

Incubate the tube at 37°C for 2 – 4 hours.
Analyze the synthesized products.

*4) The optimum concentration of DsbC is different depending on each target protein. Please use Dilution Buffer for dilution of Solution V.
We show the experiment of synthesis of functionally active proteins including disulfide bonds using PUREfrex®SS. Please visit our web site.

Note

PUREfrex®SS is developed for in vitro research only. PUREfrex®SS 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”