Purification, characterization, and specificity of dextranase inhibitor (Dei) expressed from Streptococcus sobrinus UAB108 gene cloned in Escherichia coli.
Purification, characterization, and specificity of dextranase inhibitor (Dei) expressed from Streptococcus sobrinus UAB108 gene cloned in Escherichia coli.
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大肠杆菌中克隆的远缘链球菌 UAB108 基因表达的葡聚糖酶抑制剂 (Dei) 的纯化、表征和特异性。
DOI：
10.1128/jb.177.7.1703-1711.1995
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发表时间：
1995
影响因子：
3.2
通讯作者：
Curtiss3rd,R
中科院分区：
文献类型：
--
作者：
Sun,JW;Wanda,SY;Curtiss3rd,R
来自远缘链球菌UAB 108的葡聚糖酶抑制剂基因（dei）先前被克隆、表达和测序。它的基因产物（Dei）现已被纯化为一个单一的带，表观分子量为43 kDa，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定。纯化后比活性提高了121倍。大部分Dei活性（91.2%）位于重组大肠杆菌细胞的周质部分。Dei竞争性抑制葡聚糖酶（Dex）。通过使用快速蛋白质液相色谱的凝胶过滤技术检测和回收中间体酶抑制剂（Dex-Dei）复合物，进一步证明了这种竞争性抑制机制。它们的分子量的校准表明，天然Dei作为四聚体存在，Dex作为二聚体存在，并且Dex-Dei复合物由两个Dex分子和两个Dei分子组成。缺失分析表明，完整的Dei分子是必不可少的Dei活性，但不是葡聚糖结合和免疫交叉反应。Dei是一种特殊的葡聚糖结合蛋白，具有高度特异性地抑制Dex的能力。它能抑制内源性Dex，Dex在葡糖基转移酶GTF-Ⅰ的协同作用下使葡聚糖产生更多的分支。这种抑制导致水溶性葡聚糖的积累。后一种反应产物可以抑制噬斑形成和变形链球菌细胞群的粘附。Dei来源于S. sobrinus UAB 108仅能抑制S. sobrinus（血清型d和g）、S. downei（以前的S. sobrinus，血清型h）和S.麦氏杆菌（血清型h）。这一发现表明，Dei是存在于变形链球菌群的某些血清型中的另一种重要蛋白，其通过与Dex的相互作用参与蔗糖代谢。
The dextranase inhibitor gene (dei) from Streptococcus sobrinus UAB108 was previously cloned, expressed, and sequenced. Its gene product (Dei) has now been purified as a single band with apparent molecular mass of 43 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity of Dei increased 121-fold upon purification. Most Dei activity (91.2%) was located in the periplasmic fraction from recombinant Escherichia coli cells. Dei competitively inhibits dextranase (Dex). This competitive inhibition mechanism has been further shown by detection and recovery of the intermediate enzyme-inhibitor (Dex-Dei) complex by gel filtration technology using fast protein liquid chromatography. Calibration of their molecular masses indicated that native Dei exists as a tetramer, Dex exists as dimer, and the Dex-Dei complex consists of two Dex molecules with two Dei molecules. Deletion analysis indicates that the intact Dei molecule is essential for Dei activity but not for glucan binding and immune cross-reaction. Dei is a special kind of glucan-binding protein with ability to inhibit Dex with high specificity. It can inhibit endogenous Dex, which can make more branches in glucan with the cooperation of the glucosyltransferase GTF-I. This inhibition cause the accumulation of water-soluble glucan. The latter reaction product can inhibit plaque formation and adherence of the mutans group of streptococcal cells. Dei derived from S. sobrinus UAB108 can inhibit only Dex from S. sobrinus (serotypes d and g), S. downei (previously S. sobrinus, serotype h), and S. macacae (serotype h). This finding suggests that Dei is another important protein existing in some serotypes of the mutans group of streptococci which participates in sucrose metabolism through its interaction with Dex.