Development of a Simple Protocol for Zymogram-Based Isolation and Characterization of Gingipains from Porphyromonas gingivalis: The Causative Agent of Periodontitis
Abstract
:Featured Application
Abstract
1. Introduction
2. Materials and Methods
2.1. Cultivation of P. gingivalis HG66 Strain
2.2. Preparation of Crude Protein of P. gingivalis HG66
2.3. Isolation of Gingipains Using Passive Diffusion-Mediated Gel Elution Method
2.3.1. Sample Preparation
2.3.2. Native-PAGE Electrophoresis
2.3.3. Elution of Protein
2.3.4. Gingipains Activity Assay
2.4. Characterization of Gingipains
2.4.1. SDS-PAGE
2.4.2. Gelatin Zymography
Sample Preparation
Gelatin Zymography
Gel Renaturation and Incubation
Gel Staining
3. Results
3.1. Purification: Passive-Diffusion Mediated Elution Method
3.2. Characterization of Gingipains
3.2.1. SDS-PAGE
3.2.2. Gelatin Zymography
4. Discussion
4.1. Developing and Detailing a Protocol for Isolation and Characterization of Gingipains
4.1.1. Gingipains Preparation
- 1.
- Prepare 500 mL of P. gingivalis HG66 cell-free culture supernatant by centrifuging it for 30 min at 12,000 rpm, 4 °C. Discard the pellet and preserve the supernatant on ice.
- 2.
- Add 750 mL of ice-cold acetone into the brownish supernatant slowly with vigorous agitation.
- 3.
- Obtain the protein pellet by centrifugation for 30 min at 12,000 rpm, 4 °C.
- 4.
- Resuspend the protein pellet with 10 mL of ice-cold 4,4′-dithiopyridine disulfide buffer (DTDS buffer) and incubate on ice for 1 h with agitation.
- 5.
- Transfer the protein into a dialysis bag (MWCO 12,000 to 14,000) and dialyze overnight at 4 °C with 2 changes.
- 6.
- Concentrate the protein with ultrafiltration using Amicon PM-30 membrane for 30 min at 5000 rpm, 4 °C.
- 7.
- Keep the protein sample at −20 °C for future use.
4.1.2. Isolation of Gingipains Using Passive-Diffusion Mediated Gel Elution Method
- Sample preparation
- 1.
- Transfer 100 µL of gingipains sample into the sterile 1.5 mL microcentrifuge tube.
- 2.
- Mix the gingipain sample with 20 µL of 5× non-reducing solubilizing sample buffer (without SDS and β-mercaptoethanol)
- 3.
- Load samples onto Native-polyacrylamide gel.
- Perform Gel electrophoresis
- Elution of protein
- 1.
- Cut the electrophoresed gel into 3 strips with a sterile blade.
- 2.
- Stain the protein ladder and one sample gel strip with staining buffer for 30 min with gentle agitation
- 3.
- Cut the target protein bands using the sterile blade and transfer them into the sterile 1.5 mL microcentrifuge tube.
- 4.
- Add 300 µL of ice-cold elution buffer.
- 5.
- Crush the sliced gel with a disposable homogenizer and incubate for overnight, at 4 °C.
- 6.
- Centrifuge the sample for 15 min at 10,000 rpm and collect the supernatant.
- 7.
- Test the eluted proteins with a gingipains activity assay.
- Gingipain activity assay
- 1.
- Prepare the gingipains sample of 20 µL and 50 µL for Rgp and Kgp activity assay, respectively, and dilute the samples with deionized water, making the total volume of 90 µL.
- 2.
- Add 100 µL of 2× gingipain assay buffer to the 96-wells flat bottom microtiter plates.
- 3.
- Add 90 µL of prepared gingipains sample as test group; add 90 µL of deionized water as positive control; add 100 µL of deionized water as blank.
- 4.
- Mix the samples well by pipetting them up and down gently.
- 5.
- Incubate the samples for 10 min at 37 °C.
- 6.
- Add 10 µL colorimetric substrate, Bz-L-arginine-pNA (L-BAPNA), and Ac-Lys-pNA for Rgp and Kgp activity assay respectively.
- 7.
- Measure the absorbance at 405 nm.
4.1.3. Characterization of Gingipains
SDS-PAGE
- Sample preparation
- 1.
- Transfer 50 µL of gingipains sample into the sterile 1.5 mL microcentrifuge tube.
- 2.
- Mix the gingipain sample with 10 µL of 5× non-reducing sample solubilizing buffer and 6 µL of 10% β-mercaptoethanol.
- 3.
- Boil the sample for 10 min at 100 °C.
- 4.
- Load the samples onto SDS-polyacrylamide gel.
- Perform Gel electrophoresis
- Gel staining
- 1.
- Stain the gel with Coomassie gel staining solution for 30 min at room temperature while agitating it gently.
- 2.
- De-stain the gel with the de-staining solution for 30 min until the clear and sharp, blue-colored bands are visible.
- Gel viewing
Gelatin Zymography
- Sample preparation
- 1.
- Transfer 5 µL of gingipains sample into the sterile 1.5 mL microcentrifuge tube.
- 2.
- Mix the gingipain sample with 1 µL of 5× non-reducing sample solubilizing buffer (without β-mercaptoethanol)
- 3.
- Add 200 µM of Leupeptin inhibitor (Rgp selective inhibitor), 20 µM of Cathepsin B inhibitor II (Kgp selective inhibitor), and 2 mM of TLCK (general inhibitor) into gingipains samples separately.
- 4.
- Incubate the samples for 15 min at room temperature.
- 5.
- Load samples onto SDS-gelatin-polyacrylamide gel.
- Perform Gel electrophoresis
- Gel Renaturation/Incubation
- 1.
- After electrophoresis, remove the gel from the electrophoretic gel cassette carefully.
- 2.
- Place the gel in a clean plastic container and rinse it with distilled water twice for 15 min with gentle agitation.
- 3.
- Equilibrate the gel with 100 mL of renaturation buffer for 30 min at room temperature while agitating it gently.
- 4.
- Incubate and activate the proteases present in the gel with 100 mL of incubation buffer for 2 h at 37 °C.
- Gel Staining
- 1.
- Stain the gel with Coomassie gel staining solution for 30 min at room temperature while agitating it gently.
- 2.
- De-stain the gel with de-staining buffer for 30 min until the transparent and sharp bands are observed.
- Gel viewing
4.1.4. Chemicals
4.1.5. Equipment
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Samples | Positive Control 1 | ~25 kDa 2 | ~35 kDa 2 | ~48 kDa 2 | ~63 kDa 2 | ~180 kDa 2 | ~245 kDa 2 |
---|---|---|---|---|---|---|---|
Absorbance (OD405nm) 3 | |||||||
Rgp activity | 0.073 | 0.074 | 0.064 | 0.078 | 0.075 | 0.101 | 0.073 |
Kgp activity | 0.079 | 0.085 | 0.085 | 0.061 | 0.081 | 0.091 | 0.080 |
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Wei, E.S.; Kavitha, R.; Sa’ad, M.A.; Lalitha, P.; Fuloria, N.K.; Ravichandran, M.; Fuloria, S. Development of a Simple Protocol for Zymogram-Based Isolation and Characterization of Gingipains from Porphyromonas gingivalis: The Causative Agent of Periodontitis. Appl. Sci. 2023, 13, 4314. https://doi.org/10.3390/app13074314
Wei ES, Kavitha R, Sa’ad MA, Lalitha P, Fuloria NK, Ravichandran M, Fuloria S. Development of a Simple Protocol for Zymogram-Based Isolation and Characterization of Gingipains from Porphyromonas gingivalis: The Causative Agent of Periodontitis. Applied Sciences. 2023; 13(7):4314. https://doi.org/10.3390/app13074314
Chicago/Turabian StyleWei, Eng Sze, Ramasamy Kavitha, Mohammad Auwal Sa’ad, Pattabhiraman Lalitha, Neeraj Kumar Fuloria, Manickam Ravichandran, and Shivkanya Fuloria. 2023. "Development of a Simple Protocol for Zymogram-Based Isolation and Characterization of Gingipains from Porphyromonas gingivalis: The Causative Agent of Periodontitis" Applied Sciences 13, no. 7: 4314. https://doi.org/10.3390/app13074314