Establishment of protein expression system in Porphyromonasi gingivalis

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Porphyromonas gingivalis(P.gingivalis) is a Gram-negative anaerobic bacterium and produces a number of proteinsaes. Among them, Arginine-specific cysteine proteinase(Arg-gingipain, Rgp) and lysine-specific cysteine proteinase(Lys-gingipain, Kgp) are implicated as major virulence factors in the development and progression of chronic periodontaitis. Rgp is encoded by two separate rgp genes (rgpA and rgpB), whereas Kgp is encoded by the single gene (kgp). The initial translation products of rgpA and kgp genes primarily consist of two functional parts : proteinase domain and adhesin domain. Furthermore, the C-terminal adhesin domains are derived into three or four subdomains (HGP44,HGP15,HGP17 and HGP27 for RgpA ; nHGP44,HGP15 and cHGP44 for Kgp). These C-terminal adhesin domains are highly homologous. In this study, we focused Kgp and established its expression system. Furthermore, we attempted to decide the active sites for Kgp. Although the active site of Rgp have been decided, that of Kgp was not clear due to have two cysteine residues in putative active site. Therefore, we introduced point mutation in the two putative active sites of Kgp. First, we constructed a plasmid that 248 cysteine (^<248>Cys) and 249 cysteine (^<249>Cys) were exchanged with alanine and introduced into Kgp-deficient strain KDP129. The mutation of both sites resulted in lack of emzymatic activity of Kgp, although the expression of Kgp was recognized by Western blot analysis. Next, we constructed a plasmid that point mutation plasmid against each active site and introduced into KDP129. These two mutants had the emzymatic activity of Kgp. These results suggested that ^<248>Cys and ^<249>Cys residues were important for the activity of Kgp.