Molecular analysis of protease from Treopnema denticola

采用常规色谱柱技术纯化密螺旋体denticola ATCC35405中的脯氨酸-苯丙氨酸特异性蛋白酶。从抑制剂试验中，该蛋白酶被归类为丝氨酸蛋白酶。内部蛋白酶抑制剂如抗胰蛋白酶和抗凝乳胰蛋白酶对酶活性没有影响。该酶完全降解纤维连接蛋白、抗胰蛋白酶和明胶，部分降解免疫球蛋白G重链和IV型胶原。纯化后的酶由三个多肽组成。从纯化酶中测定了nh_2末端氨基酸序列。在此基础上，对蛋白酶基因进行了克隆和测序。开放阅读框全长2169bp，编码Mr约为70kDa的蛋白。该蛋白由信号肽区、nh_2末端序列和成熟蛋白酶结构域组成。所表达的蛋白不具有蛋白水解活性，但全细胞抗体对该蛋白有反应。氨基酸序列与丝氨酸蛋白酶具有同源性。

A proline-phenylalanine specific protease from Treponema denticola ATCC35405 was purified by conventional column technique. From the inhibitor assay, this protease was classified as a serine protease. Internal protease inhibitors such as al-antitrypsin and al-antichymotrypsin have no effect on the enzymatic activity. The enzyme completely degrades fibronectin, al-antitrypsin, and gelatin and partially degrades immunoglobulin G heavy chain and type IV collagen. The purified enzyme consists of three polypeptides. NH_2-terminal amino acid sequence were determined from purified enzyme. Based on the sequences, the protease gene was cloned and sequenced. The open reading frame consists of 2,169bp and codes for a protein with a Mr of approximately 70kDa. This protein consist of signal peptide region, NH_2-terminal prosequence and mature protease domain. The expressed protein did not posessed proteolytic activity, however, the antibody against T.denticola whole cell react the protein. The deduced amino acid sequence showed its homology with a serine protease.