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人转录因子hASH4的表达纯化及其DNA结合活性

, PP. 218-224

Keywords: 螺旋-环-螺旋,碱性/螺旋-环-螺旋,hASH4,DNA结合,特异性结合,非特异性结合

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Abstract:

hASH4蛋白所属的HLH转录因子家族在调节基因表达,调控细胞周期,决定细胞分化中起了重要作用。有研究表明hASH4蛋白可能与皮肤的分化发育有着密切的关系,但具体机制不明。成功构建了pET28b-hASH4表达质粒,并在大肠杆菌BL21(DE3)中诱导表达。经过对温度、时间、IPTG浓度等表达条件的优化,确定在37℃下1mmol/LIPTG诱导表达4h可达到最佳表达效果,并通过亲和层析和弱阳离子交换层析纯化蛋白,得到了电泳纯的目的蛋白。通过非放射性凝胶滞留试验发现hASH4蛋白单体只具有非特异性的DNA结合活性,而不具有特异性的DNA结合活性,推进其在体内的转录因子功能可能需先形成异源二聚体或多聚体才能进一步特异性结合DNA进而作用于下游基因。研究结果为hASH4蛋白的功能和作用方式提供了线索,并为其进一步的结晶条件筛选、晶体结构解析和功能研究奠定基础。

References

[1]  Ma PC, Rould MA, et al. Crystal structure of MyoD bHLH domain-DNA complex:perspectives on DNA recognition and implications for transcriptional activation[J] . Cell, 1994, 77(3):451-459.
[2]  王勇, 陈克平, 姚勤. bHLH 转录因子家族研究进展[J] . 遗传, 2008, 30(7):821-830.
[3]  Hajime O, Haruki O, et al. Transcription factors involved in lens development from the preplacodal ectoderm[J] . Eev Biol, 2012, 363(2):333-347.
[4]  Massari ME, Murre C. Helix-loop-helix proteins:regulators of transcription in eucaryotic organisms [J] . Mol Cell Bio, 2000, 20(2):429-440.
[5]  Jones S. An overview of the basic helix-loop-helix proteins [J] . Genome Biol, 2004, 5(6):226-231.
[6]  Jonsson M, Mark EB, Brantsing C, et al. Hash4, a novel human achaete-scute homologue found in fetal skin[J] . Genomics, 2004, 84(5):859-866.
[7]  Michael F, Ralf M, Andreas S, et al. Translational regulation of the human achaete-scute homologue-1 by fragile X mental retardation Protein[J] . J Biol Chem, 2009, 284(7):4244-4266.
[8]  Miyamoto T, Hasuike S, Jinno Y, et al. The human ASCL2 gene escaping genomic imprinting and its expression pattern[J] . J Assist Reprod Genet, 2002, 19(5):240-244.
[9]  Huang B, Liu JY. Prokaryotic expression and purification of a cotton dehydration responsive element binding protein GhDBPl and its DNA binding activity[J] . Prog Biochem Biophys, 2006, 33(3):247-253.
[10]  Huang W, Wu QY. The ManR specifically binds to the promoter of a Nramp transporter gene in Anabaena sp. PCC 7120:a novel regulatory DNA motif in cyanobacteria[J] . Biochem Biophys Res Commun, 2004, 317(2):578-585.
[11]  Nair SK, Burley SK. X-ray structures of Myc-Max and Mad-Max recognizing DNA.Molecular bases of regulation by proto-oncogenic transcription factors[J] . Cell, 2003, 112(2):193-205.
[12]  Antonella L, Gerald PG, et al. Crystal structure of E47-NeuroD1 beta2 bHLH domain-DNA complex:heterodimer selectivity and DNA recognition[J] . Biochemistry, 2008, 47(1):218-229.
[13]  Sauv S, Tremblay L, Lavigne P. The NMR solution structure of a mutant of the Max b/HLH/LZ free of DNA:insights into the specific and reversible DNA binding mechanism of dimeric transcription factors[J] . J Mol Biol, 2004, 342(3):813-832.

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