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。这种有机体产生几种似乎是主要致病因素的蛋白酶。在本项目中，我们通过蛋白质化学和分子遗传学的方法研究了牙龈假单胞菌的蛋白水解酶的性质。我们构建了包含牙龈假单胞菌基因组片段的质粒库。对文库转化的大肠杆菌进行酪蛋白降解活性的筛选。通过对约10,000个克隆的筛选，仅获得了编码TPR蛋白酶的TPR基因。另一种筛选方法将用于克隆牙龈假单胞菌的附加蛋白水解酶基因。虽然Arg-gigiain是牙龈假单胞菌中最具特性的水解酶，但成熟蛋白在大肠杆菌中的表达尚未见报道。我们建立了精氨酸-牙周炎的表达系统。我们将使用定点突变的方法对该酶进行性质鉴定。牙龈假单胞菌的糖丙基肽酶被认为在降解宿主组织中的胶原蛋白方面起着重要作用。该酶得到完全纯化，经赖氨酰内切酶消化后，用全自动测序仪测定了部分多肽序列。我们用从多肽序列数据中推导出的寡核苷酸引物进行了聚合酶链式反应。扩增出约1,500个碱基对的核苷酸，序列分析表明该多肽属于真核细胞中的二肽基多肽酶IV。该多肽酶的编码基因已成功克隆，核苷酸全序列测定正在进行中。

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.