Purification and characterization of recombinant ligand-binding domains from the ecdysone receptors of four pest insects

Ligand binding by recombinant domains from insect ecdysone receptors

“…Assay mixtures were allowed to reach equilibrium, typically by incubating the black 96-well plate(s) overnight at 4 1C, and then at 21 1C for 2-4 h, before measuring polarization values. The small amounts of additives carried into the FP assays from the LBD heterodimer stock solutions in IMAC column elution buffer (Graham et al, 2007) did not have a significant effect on the polarization values.…”

“…Recombinant LBDs were expressed in baculovirusinfected insect cells; some experiments used recombinant cell extracts, whereas others used recombinant LBDs that had been purified by IMAC in the absence of ligand (Graham et al, 2007). Protein concentration was measured as described previously (Graham et al, 2007).…”

“…Protein concentration was measured as described previously (Graham et al, 2007). Molar concentrations of LBD heterodimer samples were calculated using the sum of the expected molecular masses for the constituent EcR and USP polypeptides, and corrected for the proportion of the population that was functional (Graham et al, 2007).…”

“…Although USP is an essential partner for receptor function, the ecdysteroid-binding pocket resides in the EcR protein (Lezzi et al, 1999). Recently, the ligandbinding domains (LBDs) of the EcR and USP proteins from different insect orders have been co-expressed recombinantly and purified by immobilized metal affinity chromatography (IMAC) and other methods (Graham et al, 2007). In addition, the atomic interactions between ligands and the LBDs of recombinant EcR-USP complexes from different insect orders have been elucidated by means of X-ray crystallography (Billas et al, 2003;Carmichael et al, 2005).…”

“…In this article, we describe the ligandbinding properties of recombinant LBDs from the ecdysone receptors of four insects that are significant agricultural pests. The species span the taxonomic orders Diptera (sheep blowfly, Lucilia cuprina), Hemiptera (peach aphid, Myzus persicae, and sweet potato whitefly, Bemisia tabaci) and Lepidoptera (cotton bollworm, Helicoverpa armigera), so the amino acid sequences of the expressed EcR DE/F segments share 80%, 57%, 60% and 61% identity, respectively, with the corresponding segment of Drosophila melanogaster EcR (Graham et al, 2007). Most of the recombinant LBD heterodimers are DE/F segments, but for H. armigera we also prepared an E/F segment to help us assess whether the D-regions contribute in any way to ligand binding.…”