Expression of ecdysteroid receptor and ultraspiracle from Chironomus tentans (Insecta, Diptera) in E. coli and purification in a functional state.
Full length clones of ecdysteroid receptor (EcR) and Ultraspiracle (USP) from Chironomus tentans were expressed as GST fusion proteins in E. coli and purified by affinity chromatography. The absence of detergents during the purification procedure is essential for retaining receptor function, especially ligand binding. Presence of USP is mandatory for ligand binding to EcR, but no other cofactors or posttranslational modifications seem to be important, since Scatchard plots revealed the same characteristics (two high affinity binding sites for Ponasterone A with K(D1)=0.24+/-0.1nM and K(D2)=3.9+/-1.3.nM) as found in 0.4 M NaCl extracts of Chironomus cells. Gel mobility shift assays showed binding of the heterodimer to PAL and DR5 even after removal of the GST-tag, whereas EcR binding to PAL1 is GST-dependent. USP binds preferentially to DR5. Addition of unprogrammed reticulocyte lysate improves ligand binding only slightly. Removal of GST has no effect on (3)H-ponasterone A binding, but alters DNA binding characteristics. Calculation of specific binding (5.3+3.0 nmol/mg GST EcR) revealed that 47+/-26% of purified receptor protein was able to bind ligand. The addition of purified EcR to cell extracts of hormone resistant subclones of the epithelial cell line from C. tentans, which have lost their ability to bind ligand, restores specific binding of (3)H-ponasterone A.
Grebe
Marco
M
Abteilung für Allgemeine Zoologie und Endokrinologie, Universität Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany.
Spindler-Barth
Margarethe
M
D000180:Acrylic Resins; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002683:Chironomidae; D002846:Chromatography, Affinity / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D029721:Drosophila Proteins; D004441:Ecdysterone / Q000031:analogs & derivatives* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D011987:Receptors, Steroid / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D000180:Acrylic Resins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C066030:USP protein, Drosophila; C034808:ecdysteroid receptor; C016680:polyacrylamide gels; D004441:Ecdysterone; C008999:ponasterone A