Purification and characterization of recombinant ligand-binding domains from the ecdysone receptors of four pest insects.
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.
Graham
Lloyd D
LD
CSIRO Molecular and Health Technologies, Sydney Laboratory, P.O. Box 184, North Ryde, NSW 1670, Australia. lloyd.graham@csiro.au
Pilling
Patricia A
PA
Eaton
Ruth E
RE
Gorman
Jeffrey J
JJ
Braybrook
Carl
C
Hannan
Garry N
GN
Pawlak-Skrzecz
Anna
A
Noyce
Leonie
L
Lovrecz
George O
GO
Lu
Louis
L
Hill
Ronald J
RJ
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
D019476:Insect Proteins; D008024:Ligands; D011987:Receptors, Steroid; D011994:Recombinant Proteins; C055469:ecdysone receptor