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Uropathogenic specific protein gene, highly distributed in extraintestinal uropathogenic Escherichia coli, encodes a new member of H-N-H nuclease superfamily

机译:尿毒症特异性蛋白基因高度分布于肠外尿毒症大肠杆菌中,编码H-N-H核酸酶超家族的新成员

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Background The uropathogenic specific protein (Usp) and three OrfU proteins (OrfU1, OrfU2 and OrfU3) are encoded in the putative small pathogenicity island which is closely associated with Uropathogenic Escherichia coli. Although homology search revealed that Usp and OrfUs have a homology with nuclease-type bacteriocins, which possess H-N-H nuclease motif, and immunity proteins respectively, the molecular activity of these proteins was never investigated. In this study, we try to over-express Usp in E. coli, purify Usp and characterize its molecular activity. Method Recombinant Usp protein was expressed in E. coli BL21(DE3) cells together with 6× Histidine tagged OrfU1 (OrfU1-His) protein, and purified with affinity chromatography using Ni2+ chelating agarose. The nuclease activity of the purified Usp was examined in vitro by using plasmid DNA as a substrate. The importance of H-N-H motif in nuclease activity of Usp was examined by site-directed mutagenesis study. Results We revealed that pET expression vector encoding Usp alone could not be maintained in E. coli BL21(DE3), and insertion of the orfUs as well as usp in the constructed plasmid diminished the toxic effect, suggesting that co-expressed OrfUs masked the activity of Usp. To purify Usp protein, we employed the expression vector encoding untagged Usp together with OrfU1-His. A tight complex formation could be observed between Usp and OrfU1-His, which allowed the purification of Usp in a single chromatographic step: binding of Usp/OrfU1-His complex to Ni2+ chelating agarose followed by elution of Usp from the complex with denaturing reagent. The purified free Usp was found to have the nuclease activity, and the activity was constitutively higher than Usp/OrfU1-His complex. H-N-H motif, which is found in various types of nucleases including a subfamily of nuclease-type bacteriocin, had been identified in the C-terminal region of Usp. Site-directed mutagenesis study showed that the H-N-H motif in Usp is indispensable for its nuclease activity. Conclusion This is the first evidence of the molecular activity of the new member of H-N-H superfamily and lays the foundation for the biological characterization of Usp and its inhibitor protein, OrfUs.
机译:背景技术尿路致病特异性蛋白(Usp)和三个OrfU蛋白(OrfU1,OrfU2和OrfU3)在与致病性大肠杆菌密切相关的假定小致病岛中编码。尽管同源性搜索显示Usp和OrfUs与分别具有H-N-H核酸酶基序的核酸酶型细菌素和免疫蛋白具有同源性,但从未研究过这些蛋白的分子活性。在这项研究中,我们试图在大肠杆菌中过表达Usp,纯化Usp并表征其分子活性。方法重组Usp蛋白与6×组氨酸标签的OrfU1(OrfU1-His)蛋白一起在大肠杆菌BL21(DE3)细胞中表达,并用Ni2 +螯合琼脂糖进行亲和色谱纯化。通过使用质粒DNA作为底物在体外检查纯化的Usp的核酸酶活性。通过定点诱变研究检查了H-N-H基序在Usp核酸酶活性中的重要性。结果我们发现,仅编码Usp的pET表达载体无法在大肠杆菌BL21(DE3)中维持,orfUs和usp插入所构建的质粒中均减弱了毒性作用,这表明共表达的OrfUs掩盖了该活性。 Usp。为了纯化Usp蛋白,我们使用了编码未标记Usp的表达载体以及OrfU1-His。在Usp和OrfU1-His之间可以观察到紧密的复合物形成,这可以在一个色谱步骤中纯化Usp:将Usp / OrfU1-His复合物与Ni2 +螯合琼脂糖结合,然后用变性剂从复合物中洗脱Usp。发现纯化的游离Usp具有核酸酶活性,并且该活性比Usp / OrfU1-His复合物组成性更高。在Usp的C末端区域中已经鉴定出在包括核酸酶型细菌素亚家族在内的各种类型的核酸酶中发现的H-N-H基序。定点诱变研究表明,Usp中的H-N-H基序对于其核酸酶活性是必不可少的。结论这是H-N-H超家族新成员分子活性的第一个证据,为Usp及其抑制剂蛋白OrfUs的生物学表征奠定了基础。

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