Expression of EcR and USP in Escherichia coli: purification and functional studies.
The functional ecdysteroid receptor complex consists of a nuclear receptor heterodimer of ecdysteroid receptor (EcR) and ultraspiracle (USP). EcR and USP of both Chironomus tentans and Drosophila melanogaster were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST). Cell lysis and protein solubilization with the anionic detergent sarkosyl yielded preparations of EcR and USP with properties similar to those of the endogenous receptors in various respects. The heterodimer of the expressed proteins specifically bound the labeled ecdysteroid (Ec) [3H]ponasterone A. Furthermore, it preferentially recognized the palindromic ecdysone response element (EcRE) PALI. Interestingly, binding to the PAL1 element was also observed for EcR homodimers. USP homodimers, in turn, preferentially bound to the direct repeat element DR1. When incubated with native polytene chromosomes of Chironomus, EcR/USP specifically accumulated at the early Ec-inducible puff site IV-2B.
Elke
C
C
Institut Für Zellbiologie, ETH-Hönggerberg, Zürich, Switzerland.
Vögtli
M
M
Rauch
P
P
Spindler-Barth
M
M
Lezzi
M
M
D000818:Animals; D002683:Chironomidae / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D004441:Ecdysterone; D004926:Escherichia coli; D007447:Invertebrate Hormones / Q000235:genetics* / Q000378:metabolism; D008024:Ligands; D011987:Receptors, Steroid / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D007447:Invertebrate Hormones; D008024:Ligands; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C066030:USP protein, Drosophila; C034808:ecdysteroid receptor; D004441:Ecdysterone; D004247:DNA