人转录因子hASH4的表达纯化及其DNA结合活性

生物技术通报 ›› 2014, Vol. 0 ›› Issue (6): 218-224.

人转录因子hASH4的表达纯化及其DNA结合活性

苏琢磊, 楼田甜, 王远东, 季朝能

（复旦大学生命科学学院遗传学研究所，上海 200433）

收稿日期:2014-04-30 出版日期:2014-06-25 发布日期:2014-06-25
作者简介:苏琢磊，男，硕士研究生，研究方向：分子遗传与结构生物学；E-mail：suzhuolei@gmail.com
基金资助:
国家自然科学基金项目（30770427），“973”课题（2009CB825505）

Expression，Purification and DNA Binding Activity of Human Transcription Factor hASH4

Su Zhuolei, Lou Tiantian, Wang Yuandong, Ji Chaoneng

（Institute of Genetics，School of Life Science，Fudan University，Shanghai 200433）

Received:2014-04-30 Published:2014-06-25 Online:2014-06-25

摘要/Abstract

摘要： 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1 mmol/L IPTG诱导表达4 h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。

关键词: 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

Abstract: hASH4 is a member of Helix-Loop-Helix（HLH）proteins which are an important group of transcription factors that exert such a determinative influence on a variety of cell proliferation, determination and differentiation from yeast to human. hASH4 has been reported closely related to skin differentiation and development, but the exact mechanism is unknown. In this study, the expression plasmid of pET28b- his- hASH4 was restructured and successfully expressed in BL21（DE3）. After the optimization of temperature, time, IPTG concentration of expression, we ascertain that 1mmol/L IPTG expressed 4 hours at 37℃ can get the best expression. and we got the electrophoretic purity of the target protein by Ni-NTA affinity chromatography and ion cation exchange chromatography. The non-radioactive EMSA experiment between DNA and protein showed that the hASH4 protein only has the non-sepcific DNA binding activity without specific DNA binding activity. The play of transcription factors by hASH4 in the body may be need to form a heterodimer or multimer to further specific binding to DNA and act on the downstream genes. This study provided clues for the really function in vivo of hASH4 and laid the foundation for the further crystallization conditions screening, structural analysis and functional studies.

Key words: HLH bHLH hASH4, DNA-binding, Specific binding, Non-specific binding

苏琢磊, 楼田甜, 王远东, 季朝能. 人转录因子hASH4的表达纯化及其DNA结合活性[J]. 生物技术通报, 2014, 0(6): 218-224.

Su Zhuolei, Lou Tiantian, Wang Yuandong, Ji Chaoneng. Expression，Purification and DNA Binding Activity of Human Transcription Factor hASH4[J]. Biotechnology Bulletin, 2014, 0(6): 218-224.

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