Establishment of protein expression system in Porphyromonasi gingivalis

牙龈卟啉单胞菌(P.givalis)是一种革兰氏阴性厌氧菌，能产生大量的蛋白质。其中，精氨酸特异性半胱氨酸蛋白酶(Arg-gigiain，RGP)和赖氨酸特异性半胱氨酸蛋白酶(Lys-gigiain，KGP)被认为是慢性牙周炎发生发展的主要毒力因子。RGP由两个独立的RGP基因(rgpA和rgpB)编码，而Kgp由单个基因(Kgp)编码。RgpA和KGP基因的初始翻译产物主要由两个功能部分组成：蛋白水解酶结构域和粘附素结构域。此外，C-末端粘附素结构域被衍生为三个或四个亚域(RgpA的HGP44、HGP15、HGP17和HGP27；KGP的nHGP44、HGP15和cHGP44)。这些C-末端的粘附素结构域高度同源。本研究以KGP为研究对象，建立了KGP的表达系统。此外，我们还试图确定KGP的活性位点。虽然RGP的活性部位已经确定，但KGP的活性部位尚不清楚，因为可能的活性部位有两个半胱氨酸残基。因此，我们在Kgp的两个可能的活性部位引入了点突变。首先，我们构建了248个半胱氨酸(^&lt；248&gt；Cys)和249个半胱氨酸(^&lt；249&gt；Cys)与丙氨酸交换的质粒，并将其导入Kgp缺陷菌KDP129。这两个位点的突变导致Kgp缺乏酶活性，但Western印迹分析证实Kgp有表达。接下来，我们构建了针对每个活性位点的点突变质粒，并将其导入KDP129。这两个突变体都具有KGP的酶活性。这些结果表明^&lt；248&gt；Cys和^&lt；249&gt；Cys残基对KGP的活性是重要的。

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.