title: Purification and characterization of recombinant ligand-binding domains from the ecdysone receptors of four pest insects.
abstract: Cloned EcR and USP cDNAs encoding the ecdysone receptors of four insect pests (Lucilia cuprina, Myzus persicae, Bemisia tabaci, Helicoverpa armigera) were manipulated to allow the co-expression of their ligand binding domains (LBDs) in insect cells using a baculovirus vector. Recombinant DE/F segment pairs (and additionally, for H. armigera, an E/F segment pair) from the EcR and USP proteins associated spontaneously with high affinity to form heterodimers that avidly bound an ecdysteroid ligand. This shows that neither ligand nor D-regions are essential for the formation of tightly associated and functional LBD heterodimers. Expression levels ranged up to 16.6mg of functional apo-LBD (i.e., unliganded LBD) heterodimer per liter of recombinant insect cell culture. Each recombinant heterodimer was affinity-purified via an oligo-histidine tag at the N-terminus of the EcR subunit, and could be purified further by ion exchange and/or gel filtration chromatography. The apo-LBD heterodimers appeared to be more easily inactivated than their ligand-containing counterparts: after purification, populations of the former were <40% active, whereas for the latter >70% could be obtained as the ligand-LBD heterodimer complex. Interestingly, we found that the amount of ligand bound by recombinant LBD heterodimer preparations could be enhanced by the non-denaturing detergent CHAPS (3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate). Purity, integrity, size and charge data are reported for the recombinant proteins under native and denaturing conditions. Certain intra- and intermolecular disulfide bonds were observed to form in the absence of reducing agents, and thiol-specific alkylation was shown to suppress this phenomenon but to introduce microheterogeneity.
authors.affiliation: CSIRO Molecular and Health Technologies, Sydney Laboratory, P.O. Box 184, North Ryde, NSW 1670, Australia. lloyd.graham@csiro.au
authors.forename: Lloyd D
authors.initials: LD
authors.lastname: Graham
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Pilling
authors.forename: Ruth E
authors.initials: RE
authors.lastname: Eaton
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Gorman
authors.forename: Carl
authors.initials: C
authors.lastname: Braybrook
authors.forename: Garry N
authors.initials: GN
authors.lastname: Hannan
authors.forename: Anna
authors.initials: A
authors.lastname: Pawlak-Skrzecz
authors.forename: Leonie
authors.initials: L
authors.lastname: Noyce
authors.forename: George O
authors.initials: GO
authors.lastname: Lovrecz
authors.forename: Louis
authors.initials: L
authors.lastname: Lu
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Hill
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004355:Drug Stability; D015870:Gene Expression; D017344:Genes, Insect; D005822:Genetic Vectors; D066298:In Vitro Techniques; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008024:Ligands; D017434:Protein Structure, Tertiary; D011987:Receptors, Steroid / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D019476:Insect Proteins; D008024:Ligands; D011987:Receptors, Steroid; D011994:Recombinant Proteins; C055469:ecdysone receptor