Molecular analysis of protease from Treopnema denticola

齿密螺旋体蛋白酶的分子分析

• 批准号: 06671833
• 负责人: ISHIHARA Kazuyuki
• 金额: $ 1.34万
• 依托单位: TOKYO DENTAL COLLEGE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06671833/
• 关键词: Treponema denticola; Serine protease; Cloning; Sequence; Pathogen; Periodontitis; Cell surface protein; Treponema dinticola; protease; cloning; sequence; pathogen

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.

采用常规柱层析技术从齿垢密螺旋体ATCC 35405中纯化了一种脯氨酸-苯丙氨酸特异性蛋白酶。从抑制剂测定，该蛋白酶被分类为丝氨酸蛋白酶。内部蛋白酶抑制剂如al-抗胰蛋白酶和al-抗胰凝乳蛋白酶对酶活性没有影响。该酶完全降解纤连蛋白、α 1-抗胰蛋白酶和明胶，并部分降解免疫球蛋白G重链和IV型胶原。纯化的酶由三种多肽组成。测定了纯化酶的氨基端氨基酸序列。在此基础上，对该蛋白酶基因进行了克隆和序列测定。开放阅读框架由2，169 bp组成，编码一个Mr约为70 kDa的蛋白。该蛋白由信号肽区、NH_2端前体序列和成熟蛋白酶结构域组成。表达的蛋白不具有蛋白水解活性，但抗齿垢毛癣菌全细胞抗体可与该蛋白发生反应。推导的氨基酸序列与丝氨酸蛋白酶同源。

项目成果

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