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Research on prokaryocyte expression and biological activity of the core region of Blooms Syndrome protein

机译:Bloom综合征蛋白核心区原核细胞的表达及生物学活性研究

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To establish an effective approach for inducing expression of the RecQ core of Bloom’s Syndrome protein (BLM642-1290) and assaying its biological activity?in vitro, BLM642-1290?recombinant protein was expressed with IPTG at room temperature inEscherichia coli, and then the expressed product was assayed using SDS-PAGE and western blotting. After purification via affinity chromatography, DNA binding activity and unwinding activity of the protein were assessed by fluorescence polarization. Furthermore, the ATPase activity of the protein was also assayed using ultraviolet spectrophotometry based on PiColorLock Gold reagent. An effective expression method was established for BLM protein in?E. coli. The obvious bioactivities of the protein were observed in binding to ssDNA or dsDNA, unwinding the dsDNA in the presence of ATP, as well as catalyzing ATP hydrolysis in the presence of ssDNA?in vitro. The prokaryocyte expression method of BLM642-1290was established successfully and the protein with biological activity was obtained from recombinant?E.coli. This would be significant to provide a better understanding on BLM protein and facilitate the elucidation of mechanism of pathopoiesia in Bloom’s Syndrome.
机译:为了建立一种诱导布鲁姆综合症蛋白(BLM642-1290)RecQ核心表达并测定其生物学活性的有效方法,将BLM642-1290重组蛋白在室温下与IPTG一起在大肠杆菌中表达,然后进行表达。使用SDS-PAGE和蛋白质印迹分析产物。通过亲和层析纯化后,通过荧光偏振评估蛋白质的DNA结合活性和解旋活性。此外,还使用基于PiColorLock Gold试剂的紫外分光光度法测定了蛋白质的ATPase活性。建立了有效的BLM蛋白在大肠杆菌中的表达方法。大肠杆菌。在与ssDNA或dsDNA结合,在ATP存在下解开dsDNA以及在ssDNA存在下催化ATP水解的过程中,观察到了该蛋白具有明显的生物活性。成功建立了BLM642-1290的原核表达方法,并从重组大肠杆菌中获得了具有生物活性的蛋白质。这将有助于更好地了解BLM蛋白,并有助于阐明Bloom综合征中的致病机理。

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