Acid-Denatured Green Fluorescent Protein (GFP) as Model Substrate to Study the Chaperone Activity of Protein Disulfide Isomerase
Abstract
:1. Introduction
2. Results and Discussion
2.1. Acid-Denatured GFP as Model Substrate
2.2. Effect of PDI Concentration
2.3. Effect of Acid-Denatured GFP Concentration
2.4. Bacitracin Inhibits the Chaperone Activity of PDI
3. Experimental Section
3.1. Vectors, Bacterial Strains, Enzymes and Chemicals
3.2. Construction of Recombinant Plasmids
3.3. Expression and Purification of GFP, YPDI and HuPDI
GFP, YPDI and HuPDI expression
GFP purification
YPDI and HuPDI purification
3.4. Acid-Denaturation of GFP
3.5. Refolding of GFP by PDI Proteins
3.6. Inhibition of the PDI Chaperone Activity by Bacitracin
3.7. Statistical and Data Analysis
4. Conclusions
Acknowledgements
References
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Spontaneous | YPDI | HuPDI | |
---|---|---|---|
k ( min−1) | 0.037 ± 0.021 | 0.107 ± 0.012 | 0.311 ± 0.027 |
Plateau (RFU) | 1.04 ± 0.48 | 1.91 ± 0.12 | 1.78 ± 0.04 |
EC50 (μM) | n.d. | 0.45 ± 0.09 | 0.34 ± 0.04 |
Hill coefficient | n.d. | 0.89 ± 0.11 | 1.06 ± 0.08 |
Kd (μM) | n.d. | 0.30 ± 0.09 | 0.18 ± 0.03 |
Primer | Sequence | Endonuclease |
---|---|---|
ScPDIF | acactcggatccCAACAAGAGGCTGTGGCC | BamHI |
ScPDIR | acactcctgcagTTACAATTCATCGTGAATGGC | PstI |
BamPDIH | cgggatccGACGCCCCCGAGGAGGAGGAC | BamHI |
HsPDIR2 | gcccaagcttACAGTTCATCTTTCACAGCTTTCTG | HindIII |
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Mares, R.E.; Meléndez-López, S.G.; Ramos, M.A. Acid-Denatured Green Fluorescent Protein (GFP) as Model Substrate to Study the Chaperone Activity of Protein Disulfide Isomerase. Int. J. Mol. Sci. 2011, 12, 4625-4636. https://doi.org/10.3390/ijms12074625
Mares RE, Meléndez-López SG, Ramos MA. Acid-Denatured Green Fluorescent Protein (GFP) as Model Substrate to Study the Chaperone Activity of Protein Disulfide Isomerase. International Journal of Molecular Sciences. 2011; 12(7):4625-4636. https://doi.org/10.3390/ijms12074625
Chicago/Turabian StyleMares, Rosa E., Samuel G. Meléndez-López, and Marco A. Ramos. 2011. "Acid-Denatured Green Fluorescent Protein (GFP) as Model Substrate to Study the Chaperone Activity of Protein Disulfide Isomerase" International Journal of Molecular Sciences 12, no. 7: 4625-4636. https://doi.org/10.3390/ijms12074625