Molecular Genetics on Proteases of Porphyromonas

牙龈卟啉单胞菌是成人牙周炎的病原菌之一。这种微生物产生几种蛋白酶，似乎是主要的毒力因子。本课题从蛋白质化学和分子遗传学的角度对牙龈卟啉单胞菌蛋白酶的性质进行了研究.我们制备了含有牙龈卟啉单胞菌基因组片段的质粒文库。用这些文库转化大肠杆菌，筛选其对酪蛋白的降解活性。在我们的筛选系统中筛选了约10，000个克隆，最终只获得了编码tpr蛋白酶的tpr基因。另一种筛选方法将用于克隆牙龈卟啉单胞菌的额外蛋白酶基因。虽然Arg-牙龈卟啉菌蛋白酶是牙龈卟啉菌蛋白酶中最具特征的蛋白酶，但成熟蛋白在大肠杆菌中的表达尚未报道。我们建立了精氨酸牙龈卟啉酶的表达系统。我们将利用定点突变的方法对该蛋白酶进行鉴定.牙龈卟啉单胞菌的糖基脯氨酰肽酶被认为在宿主组织中的胶原降解中起重要作用。该酶被完全纯化，并且在赖氨酰内肽酶消化后用自动肽测序仪测定了几个部分肽序列。我们使用从肽序列数据推导的寡核苷酸引物进行PCR方法。从该基因中扩增出约1,500个碱基，序列分析表明该肽属于真核生物二肽基肽酶IV。已成功地克隆了该肽酶的编码基因，目前正在进行核苷酸全序列测定。

Porphyromonas gingivalis is one of the etiological pathogens in adult periodontitis. This organism produces several proteases that seem to be major virulence factors. In this project, we study the properties of proteases of P.gingivalis by protein chemical and molecular genetical approaches.1. We prepared plasmid libraries containing the fragments of P.gingivalis genome. Escherichia coli transformed by these libraries, were screened for the degradation activity of casein. About 10,000 clones were screened, and only tpr gene encoding tpr protease was obtained consequently in our screening system. Another screening method will be performed for the cloning of additional protease gene of P.gingivalis.2. Although Arg-gingipain is the best characterized protease among those of P.gingivalis, expression of mature protein in E.coli has not yet been reported. We established the expression system of Arg-gingipain. We will characterize this protease by use of site-directed mutagenesis method.3. Glysylprolyl peptidase of P.gingivalis is thought to play an important role in degradation of collagen in the host tissues. This enzyme was purified completely, and several partial peptide sequences were determined with an automated peptide sequencer after lysyl endopeptidase-digestion. We performed PCR method using the oligonucleotide primers deduced from the peptide sequence data. About 1,500 base paired nucleotide was amplified, and the sequence data suggested that the peptide belongs to the group of eukaryotic dipeptidyl peptidase IV.A cloning of the gene encoding this peptidase was successfully carried out, and the determination of complete sequence of nucleotide is now in progress.