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 c omplex that mimics the native ecdysone receptor (EcR) isolated from Drosoph ila. The two steroid receptors, ultraspiracle (USP) and EcR, were expressed as truncations, retaining primarily the hormone binding domains. The recom binant receptor complex was able to mimic the pharmacology of the native re ceptor with respect to both synthetic and natural agonists. USP and EcR fus ion proteins could be expressed in separate cell lines and then recombined following isolation to yield a ligand binding preparation with a dissociati on 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 coexpress ion 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, mig rated as a variety of complexes, mostly coming out in the void volume as de natured, insoluble, aggregate. In contrast, purified extracts of coexpresse d EcR and USP eluted as a single peak with a mobility indicating a heterodi mer. The majority of the coexpressed fusion receptors, following purificati on, formed functional steroid binding sites. A detailed scheme is provided for the expression and isolation of milligram quantities of highly purified receptor dimer. (C) 1999 Academic Press.