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-hydroxyecdy sone - the key ecdysteroid controlling induction and modulation of mor phogenetic events through Drosophila development. In order to study as pects 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 e nd, we have expressed the EcR DNA-binding domain and the Usp DNA-bindi ng domain as proteins with an affinity tag consisting of six histidine residues (6xHis-EcRDBD and 6xHis-UspDBD, respectively) using the expr ession vector pQE-30. Under optimal conditions, elaborated in this stu dy, bacteria can express the recombinant 6xHis-EcRDBD to the levels of 11% of total soluble proteins and the 6xHis-UspDBD to the levels of 1 6%. Both proteins were purified to homogeneity from the soluble protei n fraction using combination of ammonium sulphate fractionation and af finity chromatography on Ni-NTA agarose. The gel mobility shift experi ments demonstrated that the purified 6xHis-EcRDBD and the 6xHis-UspDBD interact specifically with an 20-hydroxyecdysone response element fro m the promoter region of the hsp 27 Drosophila gene.