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CONTENTS
Overview
"PDI Set" is the supplement by adding to PUREfrex® series (Cell-free protein Synthesis Kits) for synthesizing proteins containing a disulfide bond in an active form.
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.
PDI Set includes oxidized glutathione (GSSG), human PDI (protein disulfide isomerase) and human Ero1α (ER oxidoreductin-1 to reoxidize PDI).
Experiment Workflow
General procedures for using PUREfrex® to synthesize functional proteins are shown in Figure.
Start by preparing the template DNA suitable to the protein synthesis with PUREfrex® and examine whether the protein of interest is synthesized. Redesign the nucleotide sequence of the template DNA if too little protein is synthesized.
Add PDI Set as supplement to PUREfrex® reaction mixture if the solubility or activity of the synthesized protein is low.
Protocols
<CASE 1> PDI and GSSG
This is a standard protocol for synthesizing proteins containing disulfide bonds using PDI Set and PUREfrex®2.1. For example, please assemble 20 µL of reaction mixture as below, in which the final concentration of each reagent is 0.5 mM Cysteine, 4 mM GSH, 1 mM GSSG and 10 µM PDI.
- 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 PDI on ice.
- Mix each solution by vortex and centrifuge briefly to collect each solution at the bottom.
- Dilute GSSG 3-fold with water.
- 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 *2 | 1 μL |
| Solution II | 1 μL |
| Solution III | 2 μL |
| PDI |
1 μL |
| Template DNA *3 | X μL |
| Total | 20 μL |
|---|
"PDI Set" is the supplement by adding to PUREfrex® for synthesizing proteins containing a disulfide bond in an active form.
- 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.
- This volume is different from Solution I of PUREfrex®2.0.
- Please use 3-fold diluted GSSG. Standard final concentration of GSSG is 1 - 3 mM with 4 mM GSH (reduced glutathione) in PUREfrex®2.1. We recommend to check the optimal concentration of GSSG because it depends on the target protein and the kind and concentration of reducing agent.
- Please visit the template DNA preparation site.
<CASE 2> PDI and Ero1α
This is a standard protocol for synthesizing proteins containing disulfide bonds using PDI Set and PUREfrex®2.1. For example, please assemble 20 µL of reaction mixture as below, in which the final concentration of each reagent is 0.5 mM Cysteine, 4 mM GSH, 10 µM PDI and 0.25 µM Ero1α.
- Thaw Solution I, Cysteine and GSH by incubation at room temperature or 37 °C for 1 minute for completely dissolving, and then cool on ice.
- Thaw Solution II, III, PDI and Ero1α 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 |
| Solution II | 1 μL |
| Solution III | 2 μL |
| 200 µM PDI | 1 μL |
| 5 µM Ero1α *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.
- This volume is different from Solution I of PUREfrex®2.0.
- Standard final concentration of PDI/Ero1α is 1/0.025 - 10/0.25 µM. We recommend to check the optimal concentration of PDI and Ero1α because it depends on the target protein. Please use attached Dilution Buffer for diluting PDI and Ero1α.
- Please visit the template DNA preparation site.
Specifications
Kit components <PDI Set>
| Reagent | Quantity for 500 μL rxn |
Quantity for 2 mL x 5 rxn |
Description | Storage *1 |
|---|---|---|---|---|
| GSSG | 25 µL | 100 µL x 5 | Oxidized glutathione (60 mM) | -20 °C |
| PDI | 25 µL | 100 µL x 5 | Human protein disulfide isomerase with no tags (200 µM) | -80 °C *2 |
| Ero1α | 25 µL | 100 µL x 5 | Human ER oxidoreductin-1 to reoxidize PDI with no tags (5 µM) | -80 °C *2 |
| Dilution buffer |
500 µL | 500 µL x 1 | 30% glycerol buffer |
-20 °C |
- Indicated temperature shows storage temperature after opening the kit. Please store the kit at -80 °C before opening.
- For storage at -80 °C, the rest of solution should be frozen rapidly in liquid nitrogen or dry ice/ethanol. Please divide into aliquots, if necessary, and avoid refreeze and thaw as much as possible.
Datasheet
PF006-0.5-EX
DownloadsPF006-10-EX
DownloadsSDS
US
DownloadsEU
DownloadsNote
PDI Set is developed for in vitro research only. PDI 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-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"
Applications
On the Resources page, you can find posters and technical notes about PUREfrex®.
Additionally, there are papers that use PUREfrex® or PURE system technology.
Related Products
When your protein needs an assistance of molecular chaperone or a formation of a disulfide bond to form a correct conformation, we have supplemental reagents by simply adding to PUREfrex® series.
Cell-free Protein Synthesis Kits
Supplements – Chaperones –
These are supplement for PUREfrex® which is molecular chaperone to be aggregate-prone protein in a soluble form.
Supplements – Forming disulfide bonds –
This is supplement for synthesizing proteins containing disulfide bonds in an active form.