Expression and purification of the hormone binding domain of the Drosophila ecdysone and ultraspiracle receptors.
Escherichia coli vectors were constructed for the production of a protein complex that mimics the native ecdysone receptor (EcR) isolated from Drosophila. The two steroid receptors, ultraspiracle (USP) and EcR, were expressed as truncations, retaining primarily the hormone binding domains. The recombinant receptor complex was able to mimic the pharmacology of the native receptor with respect to both synthetic and natural agonists. USP and EcR fusion proteins could be expressed in separate cell lines and then recombined following isolation to yield a ligand binding preparation with a dissociation constant (K(D)) for Ponasterone A of 1.5 nM and a total yield of 1.9 pmol ligand binding sites/mg protein. Alternatively, the simultaneous coexpression of both receptors increased yields by several orders of magnitude to 6 nmol ligand binding sites/mg protein with a K(D) of 0.6 nM. Chromatographic analysis under native conditions showed that EcR, when expressed alone, migrated as a variety of complexes, mostly coming out in the void volume as denatured, insoluble, aggregate. In contrast, purified extracts of coexpressed EcR and USP eluted as a single peak with a mobility indicating a heterodimer. The majority of the coexpressed fusion receptors, following purification, formed functional steroid binding sites. A detailed scheme is provided for the expression and isolation of milligram quantities of highly purified receptor dimer.
Halling
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FMC Corporation, Route 1 and Plainsboro Road, Princeton, New Jersey, 08543, USA.
Yuhas
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DA
Eldridge
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RR
Gilbey
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Deutsch
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VA
Herron
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JD
D000818:Animals; D002853:Chromatography, Liquid; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004330:Drosophila / Q000378:metabolism*; D029721:Drosophila Proteins; D004440:Ecdysone / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006639:Histidine / Q000235:genetics; D010455:Peptides / Q000235:genetics; D017434:Protein Structure, Tertiary; D011987:Receptors, Steroid / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D010455:Peptides; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C066030:USP protein, Drosophila; C055469:ecdysone receptor; C033223:polyhistidine; D004440:Ecdysone; D006639:Histidine; D013762:Tetrahydrofolate Dehydrogenase