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.
LIU Mai-li
,
YUE Xia-li
,
ZHU Qin-jun
,
YANG Yun-huang
,
WANG Hua-pu
. 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
.
DOI: 10.11938/cjmr20170101
[1] BEERLI R R, BARBAS C F. Engineering polydactyl zinc-finger transcription factors[J]. Nat Biotechnol, 2002, 20(2):135-141.
[2] CHOMETTE D, FRAIN M, CEREGHINI S, et al. Krox20 hindbrain cis-regulatory landscape:interplay between multiple long-range initiation and autoregulatory elements[J]. Development, 2006, 133(7):1253-1262.
[3] LEE S K, JURATA L W, FUNAHASHI J, et al. Analysis of embryonic motoneuron gene regulation:derepression of general activators function in concert with enhancer factors[J]. Development, 2004, 131(14):3295-3306.
[4] XUE L, CHEN X, CHANG Y, et al. Regulatory elements of the EKLF gene that direct erythroid cell-specific expression during mammalian development[J]. Blood, 2004, 103(11):4078-4083.
[5] SLUTSKY S G, KAMARAJU A K, LEVY A M, et al. Activation of myelin genes during transdifferentiation from melanoma to glial cell phenotype[J]. J Biol Chem, 2003, 278(11):8960-8968.
[6] TOMMERUP N, VISSING H. Isolation and fine mapping of 16 novel human zinc finger-encoding cdnas identify putative candidate genes for developmental and malignant disorders[J]. Genomics, 1995, 27(2):259-264.
[7] HALAMA N, YARD-BREEDIJK A, VARDARLI I, et al. The Kruppel-like zinc-finger gene ZNF236 is alternatively spliced and excluded as susceptibility gene for diabetic nephropathy[J]. Genomics, 2003, 82(3):406-411.
[8] PARLIER D, ARIZA A, CHRISTULIA F, et al. Xenopus zinc finger transcription factor IA1(Insm1) expression marks anteroventral noradrenergic neuron progenitors in Xenopus embryos[J]. Dev Dynam, 2008, 237(8):2147-2157.
[9] GIERL M S, KAROULIAS N, WENDE H, et al. The zinc-finger factor Insm1(IA-1) is essential for the development of pancreatic beta cells and intestinal endocrine cells[J]. Gene Dev, 2006, 20(17):2465-2478.
[10] BRESLIN M B, ZHU M, LAN M S. NeuroD1/E47 regulates the e-box element of a novel zinc finger transcription factor, IA-1, in developing nervous system[J]. J Biol Chem, 2003, 278(40):38991-38997.
[11] BRESLIN M B, ZHU M, NOTKINS A L, et al. Neuroendocrine differentiation factor, IA-1, is a transcriptional repressor and contains a specific DNA-binding domain:identification of consensus IA-1 binding sequence[J]. Nucleic Acids Res, 2002, 30(4):1038-1045.
[12] KAY L E, TORCHIA D A, BAX A. Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy:application to staphylococcal nuclease[J]. Biochemistry, 1989, 28(23):8972-8979.
[13] DELAGLIO F, GRZESIEK S, VUISTER G W, et al. NMRPipe:a multidimensional spectral processing system based on UNIX pipes[J]. J Biomol NMR, 1995, 6(3):277-293.
[14] GODDARD T D, KNELLER D G. SPARKY 3. University of California, San Fransisco.[J]. 2008.
[15] ROSSI P, SWAPNA G V, HUANG Y J, et al. A microscale protein NMR sample screening pipeline[J]. J Biomol NMR, 2010, 46(1):11-22.
[16] PELTON J G, TORCHIA D A, MEADOW N D, et al. Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated ⅢGlc, a signal-transducing protein from Escherichia coli, using two-dimensional heteronuclear NMR techniques[J]. Protein Sci, 1993, 2(4):543-558.
