In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.
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In This Article
Summary
Abstract
This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding1 and have a crucial role in promoting cell survival upon stress insults like heat shock2, nutrient starvation and exposure to chemicals/poisons3. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells4 as well as neurodegeneration5. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy7 and neurodegenerative disorders9. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene 11 (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45°C for 30 minutes. Upon recovery at 37°C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).
Protocol
1. Seeding the cells
- Before starting, warm up the culture medium, the PBS 1X and the trypsin in a water bath at 37˚C
- Take the cells out of the incubator and aspirate the medium.
- Gently apply 5 mL of PBS 1X on the cells to wash them.
- Aspirate the PBS 1X and apply 1 mL of trypsin containing 0,025% EDTA.
- Gently rotate the plate to have an even distribution of the trypsin.
- Place the cells in the incubator at 37˚C for 5-10 minutes (depending on the cell type). From time to time check if cells are detached by shaking the plate.
- Resuspend the cells in 10-20 mL of medium and co....
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Discussion
In this work a protocol to measure intracellular refolding activity of molecular chaperones is presented. The whole assay can be performed in 3 to 4 days as shown by the overview in Fig. 1.
The robustness and linearity of the light signal produced by the firefly and the renilla luciferase represent a solid base for the reproducibility of the protocol.
The critical step of the assay is the choice of an efficient transfection reagent to ensure the .......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
Danilo Maddalo is a recipient of a research fellowship as Young Investigator Group (YIG) from the Karlsruhe Institute of Technology.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
For this experiment a Luminometer form Perkin Elmer '1420 Luminescence Counter' Vector Light′ was used | |||
Programs for the Luminometer: | |||
Renilla: Pump 1, 100 μl injection | |||
Reaction Buffer: Pump1, 70 μl injection | |||
Luciferin: Pump2, 30μl injection | |||
For the buffers prepare these stock solutions in double distilled water: | |||
| |||
GLY-GLY-buffer: | |||
| |||
For 1 liter use 3,3g of Glycylglycin, 15ml of a 1M MgSO4 solution and 8ml of a 0,5M EGTA solution. Solve in water, adjust the pH to 7,8 and bring to a total volume of 1 liter. | |||
Reaction buffer for Renilla/C–lenterazine buffer: | |||
| |||
For 1 liter use 13,4ml of a 1M KH2PO4 solution, 86,6ml of a 1M K2HPO4 solution, 100ml of a 5M NaCl solution and 2ml of a 0,5M EDTA solution. | |||
| DMEM (Dulbecco's Modified Eagle Medium 1X) | GIBCO | 41966029 | |
| Fetal Bovine Serum Gold | PAA | A15-151 | |
| X-treme Gene 9 DNA Transfection Reagent | Roche | 06365809001 | |
| PBS Dulbecco's Phosphate Buffered Saline 1X | GIBCO | 14190-094 | |
| MOPS 3-(N-Morpholino)Propanesulfonic acid | SIGMA-ALDRICH | M1254 | |
| Cycloheximide | SIGMA-ALDRICH | C7698 | |
| Passive Lysis Buffer 5X | Promega | E194A | |
Glycylglycin EGTA MgSO4 (MgSO4*7H2O) | SIGMA-ALDRICH Roth Roth | G7278 3054.2 P027.1 | |
KH2PO4 K2HPO4 (K2HPO4*3H2O) NaCl EDTA | Roth Roth Roth Roth | 3904.1 6878.2 3957.1 8043.1 | |
| Luciferin Firefly | Biosynth | L8200 | |
| C–lenterazine | Biosynth | C7000 | |
| DTT (Dithiothreitol) | Roth | 6908.2 | |
| ATP (Adenosine Triphosphate) | Roche | 10519987001 |
References
- Bukau, B., Weissman, J., Horwich, A. Molecular chaperones and protein quality control. Cell. 125, 443-443 (2006).
- Ritossa, F. Discovery of the heat shock response. Cell Stress Chaperones. 1, 97-97 (1996).
- Hendrick, J. P., Hartl, F. U.
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