Cloning of the gene encoding glucosyltransferase from Streptococcus sobrinus

克隆、表达和测序了编码远缘链球菌(原变形链球菌6715)水不溶性葡聚糖合成(GTF-I)的糖基转移酶基因。构建远缘链霉菌6715 DNA基因库，将其构建在载体pUC18中，用抗GTF-I抗体筛选产生GTF-I多肽的克隆。分离到5个免疫阳性克隆，均产生与α-1，6-葡聚糖结合的多肽。GTF-I活性仅存在于两个大的多肽中：一个是全长的GTF-I肽，由大约1600个氨基酸残基组成(AB1克隆)，另一个是缺少约80个N-末端残基和约260个C-末端残基(AB2克隆)。对AB2克隆的缺失研究表明，GTF-I基因3‘端的缺失增加了蔗糖酶活性，失去了与水不溶性葡聚糖合成所必需的特异性葡聚糖结合。这些结果表明，GTF-I肽由三个片段组成：蔗糖裂解片段(-1,100个残基)，葡聚糖结合片段(-240个残基)，未知功能片段(-260个残基)，从N端开始。通过对AB1克隆的DNA测序发现，GTF-I多肽的一级结构与另一株远缘链霉菌的同源蛋白非常相似。

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.