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

Abstract

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

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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