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

Gingipains (RgpA, RgpB, and Kgp) are major virulence factors of the periodontitis-causing bacterium Porphyromonas gingivalis. Isolation gingipains from crude protein sample P. gingivalis is critical for studying underlying invasion mechanism that contributes to periodontitis, Alzheimer’s disease, cardiovascular disease (CVD). Chromatographic processes molecular cloning two standard techniques often used isolation, which time-consuming costly. In this study, considerably easier methods based on passive-mediated diffusion gel elution gelatin zymogram were isolate characterize gingipains. Importantly, proteins eluted Native-PAGE showed enzymatic activity both Rgp Kgp. zymography, with a size ~50 kDa above 245 suggested as arginine-specific The passive diffusion-mediated method simpler technique samples By using covalent highly specific gingipain inhibitors, zymography enabled an individual characterization inhibition. Finally, protocol can be easily extended by adding isoelectric focusing further improve separation characterization.