To generate the recombinant Porphyromonas gingivalis Gingipain R1 (RgpA), the gene coding for the RgpA protein (228-720aa) is first isolated and inserted into a plasmid vector along with the N-terminal 6xHis-tag gene. This vector is transfected into yeast cells. The yeast cells are cultured in bioreactors, where they express the RgpA protein. After sufficient growth, the cells are lysed, and the RgpA protein is purified using affinity chromatography. The purified RgpA protein undergoes quality control tests to ensure its purity. Its purity is over 90% as measured by SDS-PAGE.
Gingipain R1 (RgpA) is a 45-kDa Arg-specific cysteine proteinase of the periodontal pathogen Porphyromonas gingivalis. RgpA is a member of the gingipain family, which includes three arginine/lysine-specific proteases: RgpA, RgpB, and Kgp. These gingipains bind and cleave various host proteins, contributing to tissue destruction in periodontal diseases, which is crucial for the pathogenicity of P. gingivalis [1] [2] [3]. It is part of a polyprotein structure containing adhesin domains that aid in the bacterium's virulence [4]. Studies have shown that RgpA and RgpB are involved in the pathogen's invasion of host tissues and cause alveolar bone loss, a hallmark of periodontitis [5][6].
Furthermore, RgpA and RgpB have been shown to disrupt host defense mechanisms and contribute to tissue destruction in periodontal diseases [7]. The RgpA-Kgp proteinase-adhesin complex is crucial for the pathogen's ability to bind to and degrade host proteins, leading to periodontal bone loss [8][6]. Additionally, RgpA has been targeted in vaccine development studies as a potential strategy to induce protective immunity against P. gingivalis infections [7].
References:
[1] G. Tribble and R. Lamont, Bacterial invasion of epithelial cells and spreading in periodontal tissue, Periodontology 2000, vol. 52, no. 1, p. 68-83, 2010. https://doi.org/10.1111/j.1600-0757.2009.00323.x
[2] N. O'Brien-Simpson, P. Veith, S. Dashper, & E. Reynolds, Porphyromonas gingivalis gingipains: the molecular teeth of a microbial vampire, Current Protein and Peptide Science, vol. 4, no. 6, p. 409-426, 2003. https://doi.org/10.2174/1389203033487009
[3] R. Zhang, J. Yang, J. Wu, W. Sun, & Y. Liu, Effect of deletion of the rgpa gene on selected virulence of porphyromonas gingivalis, Journal of Dental Sciences, vol. 11, no. 3, p. 279-286, 2016. https://doi.org/10.1016/j.jds.2016.03.004
[4] R. Pathirana, N. O'Brien-Simpson, G. Brammar, N. Slakeski, & E. Reynolds, Kgp and rgpb, but not rgpa, are important for porphyromonas gingivalis virulence in the murine periodontitis model, Infection and Immunity, vol. 75, no. 3, p. 1436-1442, 2007. https://doi.org/10.1128/iai.01627-06
[5] F. Gibson and C. Genco, Prevention ofporphyromonas gingivalis-induced oral bone loss following immunization with gingipain r1, Infection and Immunity, vol. 69, no. 12, p. 7959-7963, 2001. https://doi.org/10.1128/iai.69.12.7959-7963.2001
[6] P. Rajapakse, N. O'Brien-Simpson, N. Slakeski, B. Hoffmann, & E. Reynolds, Immunization with the rgpa-kgp proteinase-adhesin complexes ofporphyromonas gingivalisprotects against periodontal bone loss in the rat periodontitis model, Infection and Immunity, vol. 70, no. 5, p. 2480-2486, 2002. https://doi.org/10.1128/iai.70.5.2480-2486.2002
[7] H. Yonezawa, K. Ishihara, & K. Okuda, Arg-gingipain a dna vaccine induces protective immunity against infection by porphyromonas gingivalis in a murine model, Infection and Immunity, vol. 69, no. 5, p. 2858-2864, 2001. https://doi.org/10.1128/iai.69.5.2858-2864.2001
[8] N. O'Brien-Simpson, R. Pathirana, R. Paolini, Y. Chen, P. Veith, V. Tamet al., An immune response directed to proteinase and adhesin functional epitopes protects against porphyromonas gingivalis-induced periodontal bone loss, The Journal of Immunology, vol. 175, no. 6, p. 3980-3989, 2005. https://doi.org/10.4049/jimmunol.175.6.3980
