蛋白质INSM1中的锌指结构域ZF (4-5)的表达、纯化与表征

doi:10.11938/cjmr20170101

波谱学杂志 ›› 2017, Vol. 34 ›› Issue (1): 1-7.doi: 10.11938/cjmr20170101

蛋白质INSM1中的锌指结构域ZF (4-5)的表达、纯化与表征

王华谱^1,2, 朱勤俊², 刘买利², 杨运煌², 岳霞丽¹

1. 华中农业大学理学院, 湖北武汉 430070;
2. 波谱与原子分子物理国家重点实验室, 武汉磁共振中心(中国科学院武汉物理与数学研究所), 湖北武汉 430071

收稿日期:2016-09-26 修回日期:2017-01-04 出版日期:2017-03-05 发布日期:2017-03-05
通讯作者: 杨运煌,Tel:027-87199541,E-mail:yang_yh@wipm.ac.cn;岳霞丽,Tel:027-87288247,E-mail:yxl@mail.hzau.edu.cn E-mail:yang_yh@wipm.ac.cn;yxl@mail.hzau.edu.cn
基金资助:
国家自然科学基金资助项目（21575155）；国家重点研发计划资助项目（2016YFA051201）

Expression,Purification and Characterization of the Zinc-Finger (4-5) Domain in Human Protein INSM1

WANG Hua-pu^1,2, ZHU Qin-jun², LIU Mai-li², YANG Yun-huang², YUE Xia-li¹

1. College of Science, Huazhong Agricultural University, Wuhan 430070, China;
2. State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan(Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences), Wuhan 430071, China

Received:2016-09-26 Revised:2017-01-04 Online:2017-03-05 Published:2017-03-05

摘要/Abstract

摘要： 胰岛素瘤相关蛋白1（INSM1）是一类转录调节蛋白，通过其C-端的锌指结构域（氨基酸250-510）来识别序列特异性的DNA分子.INSM1的C-端包含有5个串联的锌指结构域，然而这些结构域的结构及其如何识别DNA的分子机制目前仍不清楚.通过重组构建的质粒pET-32m-INSM1（424-497）表达的蛋白质（氨基酸424-497）包含了最后两个锌指结构域4和5，简称为ZF（4-5）.该文详细研究了蛋白质ZF（4-5）的诱导表达条件，得到了较高产率的纯化蛋白.核磁共振（NMR）谱和圆二色谱（CD）揭示了Zn²⁺对稳定锌指蛋白结构的必要性，以及C2H2-Zn²⁺结合的组氨酸呈现为δ-异构方式.

关键词: 液体核磁共振(solution NMR), 结构与功能, 锌指蛋白, 表达与纯化

Abstract: Human insulinoma-associated protein 1 (INSM1) is a transcriptional regulator recognizing sequence-specific DNA through its C-terminal zinc finger (ZF) domains. INSM1 contains five zinc finger domains, whose structures are still not known; therefore, the mechanisms through which it recognizes DNA also remain unclear. In this study, we designed the recombinant plasmid pET-32m-INSM1(424-497), which can express the truncated INSM1 fragment containing the zinc finger domains 4 and 5[i.e., ZF(4-5)]. Expression and purification of ZF(4-5) were explored in order to achieve high protein yield for further structural and functional study. Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectra revealed that chelation of Zn²⁺ to C2H2 in the ZF(4-5) was important for its structural stability, and also confirmed that the active-sites, Zn²⁺-chelated histidines, had the δ-tautomeric form.

Key words: solution NMR, protein expression and purification, structure and function, zinc finger domain

中图分类号:

O482.53

王华谱, 朱勤俊, 刘买利, 杨运煌, 岳霞丽. 蛋白质INSM1中的锌指结构域ZF (4-5)的表达、纯化与表征[J]. 波谱学杂志, 2017, 34(1): 1-7.

WANG Hua-pu, ZHU Qin-jun, LIU Mai-li, YANG Yun-huang, YUE Xia-li. Expression,Purification and Characterization of the Zinc-Finger (4-5) Domain in Human Protein INSM1[J]. Chinese Journal of Magnetic Resonance, 2017, 34(1): 1-7.

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蛋白质INSM1中的锌指结构域ZF (4-5)的表达、纯化与表征

Expression,Purification and Characterization of the Zinc-Finger (4-5) Domain in Human Protein INSM1

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蛋白质INSM1中的锌指结构域ZF (4-5)的表达、纯化与表征

Expression,Purification and Characterization of the Zinc-Finger (4-5) Domain in Human Protein INSM1

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PSSS₅₀-b-PNIPAM₃₀₀嵌段共聚物在二元溶剂中自组装的NMR研究