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首页> 外文期刊>Journal of synchrotron radiation >Crystal structure analysis, overexpression and refolding behaviour of a DING protein with single mutation
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Crystal structure analysis, overexpression and refolding behaviour of a DING protein with single mutation

机译:单个突变的DING蛋白的晶体结构分析,过表达和重折叠行为

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After crystallization of a certain protein-RNA complex, well diffracting crystals were obtained. However, the asymmetric unit of the crystal was too small to locate any components. Mass spectrometry and X-ray crystal structure analysis showed that it was a member of the DING protein family (HPBP). Surprisingly, the structure of HPBP reported previously was also determined accidentally as a contaminant, suggesting that HPBP has a strong tendency to crystallize. Furthermore, DING proteins were reported to relate in disease. These observations suggest that DING has potential for application in a wide range of research fields. To enable further analyses, a system for preparation of HPBP was constructed. As HPBP was expressed in insoluble form in Escherichia coli, it was unfolded chemically and refolded. Finally, a very high yield preparation method was constructed, in which 43 mg of HPBP was obtained from 1 L of culture. Furthermore, to evaluate the validity of refolding, its crystal structure was determined at 1.03 ? resolution. The determined structure was identical to the native structure, in which two disulfide bonds were recovered correctly and a phosphate ion was captured. Based on these results, it was concluded that the refolded HPBP recovers its structure correctly.
机译:在某种蛋白质-RNA复合物结晶后,获得了良好衍射的晶体。但是,晶体的不对称单元太小,无法定位任何组分。质谱和X射线晶体结构分析表明它是DING蛋白家族(HPBP)的成员。出人意料的是,先前报道的HPBP的结构也被偶然地确定为污染物,这表明HPBP具有很强的结晶趋势。此外,据报道DING蛋白与疾病有关。这些观察结果表明,DING在许多研究领域中都有应用潜力。为了能够进行进一步的分析,构建了用于制备HPBP的系统。由于HPBP以不溶形式在大肠杆菌中表达,因此它在化学上展开并重新折叠。最后,构建了一种非常高产量的制备方法,其中从1 L培养物中获得了43 mg HPBP。此外,为了评价重折叠的有效性,将其晶体结构确定为1.03≤m。解析度。所确定的结构与天然结构相同,在天然结构中,正确地回收了两个二硫键并捕获了磷酸根离子。根据这些结果,可以得出结论,重新折叠的HPBP正确地恢复了其结构。

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