Cloning of the gene encoding glucosyltransferase from Streptococcus sobrinus

远缘链球菌葡萄糖基转移酶编码基因的克隆

• 批准号: 63570871
• 负责人: FUKUI Kazuhiro
• 金额: $ 1.34万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63570871/
• 关键词: Streptococcus sobrinus; glucosyltransferase gene; cloning; water-insoluble glucan; dextran binding domain; Streptococcus mutans; 不溶性グネカン合成酵素; グルコシ-ルトランスフェレ-ス; クロ-ニング; ショ糖; スクラ-ゼ; 不溶性グルカン合成酵素; グルコシールトランスフェレース遺伝子のクローニング; 蔗糖; スクラーゼ

The gene encoding glucosyltransferase responsible for water-insoluble glucansynthesis (GTF-I) of streptococcus sobrinus(formerly Streptococcus mutans 6715) was cloned, expressed, and sequenced. A gene bank from S.sobrinus 6715 DNA was contructed in vector pUC18 and screened with anti-GTF-I antibody to detect clones producing GTF-I peptide. Five immunopositive clones were isolated, all of which produced peptides that bound alpha-1,6 glucan.GTF-I activity was found in only two large peptides : one stretching over the full length of the GTF-I peptide and composed of about 1,600 amino acid residues (AB1 clone) and the other lacking about 80 N-terminal residues and about 260 C-terminal residues (AB2 clone). A deletion study of the AB2 clone indicated that specific glucan binding, which is essential for water-insoluble glucan synthesis, was lost prior to sucrase activity with an increase in deletion from the 3'end of the GTF-I gene.These results suggest that the GTF-I peptide consists of three segments : that for sucrose splitting (-1,100 residues), that for glucan binding (-240 residues), and that unknown function (-260 residues), in order from the N terminus. The primary structure of GTF-I peptide, deduced by DNA sequencing of the AB1 clone, was found to be very similar to that of the homologus protein from another strain of S.sobrinus.

对变形链球菌（Streptococcus mutans 6715）的葡萄糖基转移酶基因进行了克隆、表达和测序。以远缘链球菌6715 DNA为模板构建基因库，用抗GTF-Ⅰ抗体筛选产生GTF-Ⅰ肽的克隆。分离出5个免疫阳性克隆，它们都产生结合α-1，6葡聚糖的肽，仅在两个大肽中发现GTF-Ⅰ活性：一个延伸至GTF-Ⅰ肽的全长，由约1，600个氨基酸残基组成（AB 1克隆），另一个缺少约80个N-末端残基和约260个C-末端残基（AB 2克隆）。AB 2克隆的缺失研究表明，特异性葡聚糖结合是水不溶性葡聚糖合成所必需的，在蔗糖酶活性之前丧失，从GTF-1基因3 '端缺失增加。从N端开始依次为蔗糖裂解（~ 1，100个残基）、葡聚糖结合（~ 240个残基）和未知功能（~ 260个残基）。GTF-I肽的一级结构，推导出的AB 1克隆的DNA测序，被发现是非常相似的同源蛋白质从另一株S. sobrinus。

项目成果

Hirokazu Abo: "Poptide Sequences Sucrose Splitting and Glucan Binding within Stroptcoccus sobrinus Glucosyltansforase(Water-Insoluble Glucan Synthetase)" Journal of Bactoriology. 173. 989-996 (1991)

Hirokazu Abo：“链球菌葡糖基聚糖合成酶（水不溶性葡聚糖合成酶）内的蔗糖裂解和葡聚糖结合的肽序列”细菌学杂志。

Hirokazu ABO: "Peptide Sequences Sucrose Splitting and Glucan Binding within Streptococcus sobrinus Glucosyltransferase(Water-Insoluble Gulucan Synthetase)" Journal of Bacteriology. 173. 989-996 (1991)

Hirokazu ABO：“链球菌葡糖基转移酶（水不溶性葡聚糖合成酶）内蔗糖裂解和葡聚糖结合的肽序列”细菌学杂志。