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| NR4A1 DNA 结合域的表达纯化 | |
| 引用本文： | 闫宁宁,李俊,陈小娟,陈永恒,陈林,陈主初.NR4A1 DNA 结合域的表达纯化[J].中南大学学报(医学版),2015,40(4):345-350. |
| 作者姓名： | 闫宁宁 李俊 陈小娟 陈永恒 陈林 陈主初 |
| 作者单位： | 1. 中南大学湘雅医院卫生部肿瘤蛋白质组学重点实验室结构生物学实验室，长沙 410008； 2. 南加州大学化学与生命科学系，美国 洛杉矶 90089 |
| 基金项目： | 国家科学自然基金(81272971).This work was supported by the National Natural Science Foundation of China |
| 摘 要： | 目的：寻求一种核受体家族蛋白NR4A1 DNA结合域(NR4A1 DNA binding domain，NR4A1-DBD)表达纯化的
方法。方法：构建融合蛋白PET28a-NR4A1-DBD表达载体，分别通过镍亲和层析、阳离子交换层析及凝胶过滤的方法
对目的蛋白进行纯化。结果：PET28a-NR4A1-DBD蛋白在低温诱导(24 ℃)时多以可溶性形式存在，通过此纯化，可达
到每升培养基产生2~3 mg蛋白，纯度高达95%以上。结论：镍亲和层析是一种可靠、实用的蛋白表达纯化方法，后续
使用阳离子交换层析及凝胶过滤可以进一步提高蛋白纯度。 |
| 关 键 词： | NR4A1 层析 阳离子交换 表达 纯化 |
Expression and purification of DNA binding domain of NR4A1 |
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| YAN Ningning,LI Jun,CHEN Xiaojuan,CHEN Yongheng,CHEN Lin,CHEN Zhuchu.Expression and purification of DNA binding domain of NR4A1[J].Journal of Central South University (Medical Sciences)Journal of Central South University (Medical Sciences),2015,40(4):345-350. | |
| Authors: | YAN Ningning LI Jun CHEN Xiaojuan CHEN Yongheng CHEN Lin CHEN Zhuchu |
| Institution: | 1. Laboratory of Structural Biology, Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, China; 2. Departments of Chemistry and Biological Sciences, University of Southern California, Los Angeles CA 90089, USA |
| Abstract: | Objective: To express and purify NR4A1-DNA binding domain (DBD) protein of nuclear receptors. Methods: Th e fusion protein PET28a-NR4A1-DBD was constructed and purifi ed with the nickel affi nity chromatography, cation-exchange chromatography and gel fi ltration chromatography. Results: Th e protein PET28a-NR4A1-DBD was mostly soluable at 24 ℃. A total of 2–3 mg/L pure NR4A1 proteins were yielded in bacterial culture and the purity for fi nal fractions of NR4A1-DBD protein were great than 95% by SDS-PAGE analysis. Conclusion: Nickel affi nity chromatography is eff ective to purify protein. Th e protein purity can be further improved by the following methods including cation-exchange chromatography and gel filtration chromatography. |
| Keywords: | NR4A1 chromatography cation-exchange expression purification |
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