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Development of a Simple Protocol for Zymogram-Based Isolation and Characterization of Gingipains from Porphyromonas gingivalis: The Causative Agent of Periodontitis

Authors :
Eng Sze Wei
Ramasamy Kavitha
Mohammad Auwal Sa’ad
Pattabhiraman Lalitha
Neeraj Kumar Fuloria
Manickam Ravichandran
Shivkanya Fuloria
Source :
Applied Sciences, Vol 13, Iss 7, p 4314 (2023)
Publication Year :
2023
Publisher :
MDPI AG, 2023.

Abstract

Gingipains (RgpA, RgpB, and Kgp) are major virulence factors of the periodontitis-causing bacterium Porphyromonas gingivalis. Isolation of gingipains from the crude protein sample of P. gingivalis 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 P. gingivalis. 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.

Details

Language :
English
ISSN :
20763417
Volume :
13
Issue :
7
Database :
Directory of Open Access Journals
Journal :
Applied Sciences
Publication Type :
Academic Journal
Accession number :
edsdoj.8b32f60ff224845ac8b854266eaab1f
Document Type :
article
Full Text :
https://doi.org/10.3390/app13074314