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首页> 外文期刊>Infection and immunity >Purification and partial characterization of the multicomponent dextranase complex of Streptococcus sobrinus and cloning of the dextranase gene.
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Purification and partial characterization of the multicomponent dextranase complex of Streptococcus sobrinus and cloning of the dextranase gene.

机译：链球菌多组分葡聚糖酶复合物的纯化和部分表征，以及葡聚糖酶基因的克隆。

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The presence of proteases in culture supernatant fluids and on the cell surface of Streptococcus sobrinus and the aggregation of multicomponent enzyme complexes make the isolation and characterization of cell surface proteins difficult. We report a simple purification procedure for dextranase and the cloning of the dextranase structural gene. S. sobrinus culture supernatant fluids were precipitated with 70% ammonium sulfate, and the precipitate was dialyzed against sodium acetate buffer and loaded onto a hemoglobin-Sepharose 4B column connected to a blue dextran-agarose column at 4 degrees C. After being washed with low concentrations of salt, the dextranase and the dextran-binding proteins were eluted with 5 M KI and further purified by gel filtration. Two dextranases (molecular weights, 175,000 and 160,000) were purified and partially characterized. The structural gene for the dextranase of S. sobrinus 6715 strain UAB66, serotype g, was cloned into the cosmid vector, pHC79. Clones were selected for expression of dextranase activity by detection of zones of enzyme-mediated hydrolysis of a blue dextran substrate incorporated into minimal medium agar plates. Release of dextranase was achieved by induction of thermoinducible, excision-defective Escherichia coli K-12 lysogens containing recombinant cosmid molecules of S. sobrinus DNA. Recombinant cosmid molecules were repackaged simultaneously into infectious lambdoid particles. Recombinant clones expressing dextranase activity which varied in size from the high-molecular-weight protein produced by S. sobrinus (i.e., 175,000) to lower-molecular-weight forms expressed by S. sobrinus have been identified and partially characterized.
机译：蛋白酶在培养上清液中和链球菌的细胞表面上的存在以及多组分酶复合物的聚集使细胞表面蛋白的分离和鉴定变得困难。我们报告了一个简单的葡聚糖酶纯化程序和葡聚糖酶结构基因的克隆。用70％硫酸铵沉淀Sobrinus培养物上清液，并将沉淀物用乙酸钠缓冲液透析，并在4°C加载到连接到蓝色葡聚糖-琼脂糖柱的血红蛋白-Sepharose 4B柱上。浓度的盐，葡聚糖酶和葡聚糖结合蛋白用5 M KI洗脱，并通过凝胶过滤进一步纯化。纯化并部分表征了两种葡聚糖酶（分子量分别为175,000和160,000）。将S. sobrinus 6715菌株UAB66的葡聚糖酶的结构基因（血清型g）克隆到粘粒载体pHC79中。通过检测掺入基本培养基琼脂平板中的蓝色葡聚糖底物的酶介导的水解区域来选择表达葡聚糖酶活性的克隆。葡聚糖酶的释放是通过诱导热诱导的，切除缺陷的大肠杆菌K-12溶原菌而实现的，所述溶菌原含有so.sbrinus DNA的重组粘粒分子。重组粘粒分子同时被重新包装成传染性lambdoid颗粒。已经鉴定并部分表征了表达葡聚糖酶活性的重组克隆，其大小从由链球菌产生的高分子量蛋白质（即175,000）到由链球菌表达的较低分子量形式而变化。

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