title: GST-Induced dimerization of DNA-binding domains alters characteristics of their interaction with DNA.
abstract: The steroid hormone 20-hydroxyecdysone (20E) plays a key role in the induction and modulation of morphogenetic events throughout Drosophila melanogaster development. Two members of the nuclear receptor superfamily, the product of the EcR (EcR) and of the ultraspiracle genes (Usp), heterodimerize to form its functional receptor. To study the receptor-DNA interaction, critical for regulating 20E-dependent gene expression, it is necessary to produce large quantities of EcR and Usp DNA-binding domains. Toward this end DNA-binding domains of EcR and Usp (EcRDBD and UspDBD, respectively) were cloned and expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST). However, the results of DNA-binding studies obtained with purified GST-DBDs were found to be questionable because the fused proteins oligomerized in solution due to the presence of GST. Therefore DBDs were released from GST-chimeric proteins by thrombin cleavage and then purified by glutathione-Sepharose 4B chromatography and by gel filtration on Superdex 75 HR. The gel mobility-shift experiments showed that UspDBD exhibited higher affinity than EcRDBD toward a 20-hydroxyecdysone response element from the Drosophila hsp 27 gene (hsp 27pal). Furthermore, formation of the heterodimeric EcRDBD-UspDBD complex was observed to be synergistic when equimolar mixture of both DBDs was incubated with hsp 27pal. Surprisingly, GST-EcRDBD bound hsp 27pal with higher affinity than GST-UspDBD. This difference was accompanied by the impaired ability of the GST-DBDs to interact synergistically with hsp 27pal. This is the first report on expression and purification of the soluble DBDs of the functional ecdysteroid receptor with satisfying yields. Furthermore, our results add to the recent findings which indicate the need for caution in interpreting the activities of GST fusion proteins.
authors.affiliation: Biochemistry and Biotechnology, Division of Biochemistry, Wroclaw University of Technology, Wybrzeze Wyspiańskiego 27, Wroclaw, 50-370, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Niedziela-Majka
authors.forename: G
authors.initials: G
authors.lastname: Rymarczyk
authors.forename: M
authors.initials: M
authors.lastname: Kochman
authors.forename: A
authors.initials: A
authors.lastname: Ozyhar
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000502:physiology; D004441:Ecdysterone / Q000502:physiology*; D004926:Escherichia coli; D017344:Genes, Insect / Q000235:genetics; D006360:Heat-Shock Proteins / Q000737:chemistry*; D019476:Insect Proteins / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry; D011987:Receptors, Steroid / Q000737:chemistry*; D011994:Recombinant Proteins / Q000302:isolation & purification; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D006360:Heat-Shock Proteins; D019476:Insect Proteins; D009838:Oligodeoxyribonucleotides; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D014157:Transcription Factors; C066030:USP protein, Drosophila; C034808:ecdysteroid receptor; D004441:Ecdysterone