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

doi:10.11938/cjmr20170101

Chinese Journal of Magnetic Resonance ›› 2017, Vol. 34 ›› Issue (1): 1-7.doi: 10.11938/cjmr20170101

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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

Abstract

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

CLC Number:

O482.53

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

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