Development of a Simple Protocol for Zymogram-Based Isolation and Characterization of Gingipains from <i>Porphyromonas gingivalis</i>: The Causative Agent of Periodontitis
Gingipains (RgpA, RgpB, and Kgp) are major virulence factors of the periodontitis-causing bacterium <i>Porphyromonas gingivalis</i>. Isolation of gingipains from the crude protein sample of <i>P. gingivalis</i> is critical for studying the underlying invasion mechanism that c...
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MDPI AG
2023-03-01
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| author | Eng Sze Wei Ramasamy Kavitha Mohammad Auwal Sa’ad Pattabhiraman Lalitha Neeraj Kumar Fuloria Manickam Ravichandran Shivkanya Fuloria |
| author_facet | Eng Sze Wei Ramasamy Kavitha Mohammad Auwal Sa’ad Pattabhiraman Lalitha Neeraj Kumar Fuloria Manickam Ravichandran Shivkanya Fuloria |
| author_sort | Eng Sze Wei |
| collection | DOAJ |
| description | Gingipains (RgpA, RgpB, and Kgp) are major virulence factors of the periodontitis-causing bacterium <i>Porphyromonas gingivalis</i>. Isolation of gingipains from the crude protein sample of <i>P. gingivalis</i> is critical for studying the underlying invasion mechanism that contributes to periodontitis, Alzheimer’s disease, and cardiovascular disease (CVD). Chromatographic processes and molecular cloning are two standard techniques often used for gingipains isolation, which are time-consuming and costly. In this study, considerably easier methods based on passive-mediated diffusion gel elution and gelatin zymogram were used to isolate and characterize gingipains. Importantly, proteins eluted from Native-PAGE showed enzymatic activity for both Rgp and Kgp. In gelatin zymography, the proteins with a molecular size of ~50 kDa and above 245 kDa were suggested as arginine-specific gingipains. The passive diffusion-mediated gel elution method is a simpler technique to isolate gingipains from crude protein samples of <i>P. gingivalis</i>. By using covalent and highly specific gingipain inhibitors, gelatin zymography enabled an individual characterization of gingipain activity and inhibition. Finally, this protocol can be easily extended by adding the isoelectric focusing to further improve the protein separation and characterization. |
| first_indexed | 2024-03-11T05:43:18Z |
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| issn | 2076-3417 |
| language | English |
| last_indexed | 2024-03-11T05:43:18Z |
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| spelling | doaj.art-d8b32f60ff224845ac8b854266eaab1f2023-11-17T16:18:34ZengMDPI AGApplied Sciences2076-34172023-03-01137431410.3390/app13074314Development of a Simple Protocol for Zymogram-Based Isolation and Characterization of Gingipains from <i>Porphyromonas gingivalis</i>: The Causative Agent of PeriodontitisEng Sze Wei0Ramasamy Kavitha1Mohammad Auwal Sa’ad2Pattabhiraman Lalitha3Neeraj Kumar Fuloria4Manickam Ravichandran5Shivkanya Fuloria6Department of Biotechnology, Faculty of Applied Science, AIMST University, Bedong 08100, Kedah, MalaysiaDepartment of Biotechnology, Faculty of Applied Science, AIMST University, Bedong 08100, Kedah, MalaysiaDepartment of Biotechnology, Faculty of Applied Science, AIMST University, Bedong 08100, Kedah, MalaysiaDepartment of Biochemistry, Faculty of Medicine, AIMST University, Bedong 08100, Kedah, MalaysiaFaculty of Pharmacy, AIMST University, Bedong 08100, Kedah, MalaysiaDepartment of Biotechnology, Faculty of Applied Science, AIMST University, Bedong 08100, Kedah, MalaysiaFaculty of Pharmacy, AIMST University, Bedong 08100, Kedah, MalaysiaGingipains (RgpA, RgpB, and Kgp) are major virulence factors of the periodontitis-causing bacterium <i>Porphyromonas gingivalis</i>. Isolation of gingipains from the crude protein sample of <i>P. gingivalis</i> is critical for studying the underlying invasion mechanism that contributes to periodontitis, Alzheimer’s disease, and cardiovascular disease (CVD). Chromatographic processes and molecular cloning are two standard techniques often used for gingipains isolation, which are time-consuming and costly. In this study, considerably easier methods based on passive-mediated diffusion gel elution and gelatin zymogram were used to isolate and characterize gingipains. Importantly, proteins eluted from Native-PAGE showed enzymatic activity for both Rgp and Kgp. In gelatin zymography, the proteins with a molecular size of ~50 kDa and above 245 kDa were suggested as arginine-specific gingipains. The passive diffusion-mediated gel elution method is a simpler technique to isolate gingipains from crude protein samples of <i>P. gingivalis</i>. By using covalent and highly specific gingipain inhibitors, gelatin zymography enabled an individual characterization of gingipain activity and inhibition. Finally, this protocol can be easily extended by adding the isoelectric focusing to further improve the protein separation and characterization.https://www.mdpi.com/2076-3417/13/7/4314isolationcharacterizationzymographygingipains<i>Porphyromonas gingivalis</i>Rgp |
| spellingShingle | Eng Sze Wei Ramasamy Kavitha Mohammad Auwal Sa’ad Pattabhiraman Lalitha Neeraj Kumar Fuloria Manickam Ravichandran Shivkanya Fuloria Development of a Simple Protocol for Zymogram-Based Isolation and Characterization of Gingipains from <i>Porphyromonas gingivalis</i>: The Causative Agent of Periodontitis Applied Sciences isolation characterization zymography gingipains <i>Porphyromonas gingivalis</i> Rgp |
| title | Development of a Simple Protocol for Zymogram-Based Isolation and Characterization of Gingipains from <i>Porphyromonas gingivalis</i>: The Causative Agent of Periodontitis |
| title_full | Development of a Simple Protocol for Zymogram-Based Isolation and Characterization of Gingipains from <i>Porphyromonas gingivalis</i>: The Causative Agent of Periodontitis |
| title_fullStr | Development of a Simple Protocol for Zymogram-Based Isolation and Characterization of Gingipains from <i>Porphyromonas gingivalis</i>: The Causative Agent of Periodontitis |
| title_full_unstemmed | Development of a Simple Protocol for Zymogram-Based Isolation and Characterization of Gingipains from <i>Porphyromonas gingivalis</i>: The Causative Agent of Periodontitis |
| title_short | Development of a Simple Protocol for Zymogram-Based Isolation and Characterization of Gingipains from <i>Porphyromonas gingivalis</i>: The Causative Agent of Periodontitis |
| title_sort | development of a simple protocol for zymogram based isolation and characterization of gingipains from i porphyromonas gingivalis i the causative agent of periodontitis |
| topic | isolation characterization zymography gingipains <i>Porphyromonas gingivalis</i> Rgp |
| url | https://www.mdpi.com/2076-3417/13/7/4314 |
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