Expression and purification of 6xHis-tagged DNA binding domains of functional ecdysteroid receptor from drosophila melanogaster.
Two members of the nuclear receptor superfamily, EcR and Ultraspiracle (Usp) heterodimerize to form a functional receptor for 20-hydroxyecdysone-the key ecdysteroid controlling induction and modulation of morphogenetic events through Drosophila development. In order to study aspects of receptor function and ultimately the structural basis of the ecdysteroid receptor-DNA interaction, it is necessary to produce large quantities of purified EcR and Usp DNA-binding domains. Toward this end, we have expressed the EcR DNA-binding domain and the Usp DNA-binding domain as proteins with an affinity tag consisting of six histidine residues (6xHis-EcRDBD and 6xHis-UspDBD, respectively) using the expression vector pQE-30. Under optimal conditions, elaborated in this study, bacteria can express the recombinant 6xHis-EcRDBD to the levels of 11% of total soluble proteins and the 6xHis-UspDBD to the levels of 16%. Both proteins were purified to homogeneity from the soluble protein fraction using combination of ammonium sulphate fractionation and affinity chromatography on Ni-NTA agarose. The gel mobility shift experiments demonstrated that the purified 6xHis-EcRDBD and the 6xHis-UspDBD interact specifically with an 20-hydroxyecdysone response element from the promoter region of the hsp 27 Drosophila gene.
Rusin
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A
Institute of Organic Chemistry, Biochemistry and Biotechnology, Technical University of Wrocław, Poland.
Niedziela-Majka
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A
Rymarczyk
G
G
Ozyhar
A
A
D000818:Animals; D002847:Chromatography, Agarose; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004331:Drosophila melanogaster; D004926:Escherichia coli / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics; D006639:Histidine / Q000378:metabolism*; D011987:Receptors, Steroid / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D011987:Receptors, Steroid; C034808:ecdysteroid receptor; D006639:Histidine