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

Featured Application: Porphyromonas gingivalis is an oral bacterium that uses gingipains as its primary invasion factor to cause periodontitis and certain systemic diseases in humans. To study the underlying mechanisms employed by gingipains, a simpler and quick approach is required to examine the protease activity. In the present study, passive diffusion-mediated protein elution and gelatin zymography methods were demonstrated to isolate and characterize the gingipains. This study found that an eluted protein with a molecular weight of about 180 kDa exhibited both Rgp and Kgp activity. While in the gelatin zymography, the proteins with a molecular weight of approximately 50 kDa, and above 245 kDa were found to be arginine-X gingipains when specific selective inhibitors, Leupeptin and Cathepsin B inhibitor II were used. The present study protocol will allow a more efficient separation of targeted protein samples with a greater difference in isoelectric point. 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. [ABSTRACT FROM AUTHOR]