目的:建立CUEDC2中CUE结构域的原核表达系统,获得13C、15N同位素标记的CUE结构域蛋白质,以用于结构生物学研究.方法:利用分子生物学方法将CUE结构域编码序列构建到pET-28a原核表达系统,表达和纯化13C、15N标记的重组蛋白;用SDS-PAGE等方法对其进行鉴定.结果:目的蛋白经SDS-PAGE和MALDI-TOF/MS检测,相对分子质量正确,圆二色谱和核磁共振波谱结果显示目的蛋白折叠良好.结论:获得了高浓度、高纯度、折叠良好的CUE结构域标记蛋白质,利于进一步的结构生物学研究.%Objective: To obtain stable-isotope 13C/15N-labeled CUE domain of CUEDC2 by constructing prokaryotic expression vector carrying DNA fragment encoding CUE domain in CUEDC2 for structural and functional studies. Methods: The coding sequence of the CUE was cloned into pET-28a prokaryotic expression system. The His-tagged CUE was expressed and purified with affinity purification technology, identified with SDS-PAGE, MALDI-TOF/MS, circular dichroism and NMR. Results: The target peptide was correctly expressed as indicated by the results from SDS-PAGE and MALDI-TOF/MS; 2D 1H-15N HSQC NMR spectroscopy and circular dichroism data showed that CUE domain was well folded. Conclusion: Well folded CUE domain sample with isotope-labeling was correctly expressed with high quality and was suitable for further solution structure study.
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