Preparation of functional ecdysteroid receptor proteins (EcR and USP) using a wheat germ cell-free protein synthesis system

Abstract

Ecdysone receptor (EcR) and ultraspiracle (USP) of the lepidopteran Chilo suppressalis were expressed using a wheat germ cell-free protein synthesis system, and the functions of the expressed proteins were analyzed. In a gel mobility shift assay, the EcR/USP heterodimer bound to the Drosophila melanogaster hsp27 ecdysone response element. In ligand-receptor binding assays, a potent ecdysteroid, ponasterone A, bound to the EcR/USP complex with a high affinity (KD=2.1 nM). For a series of ecdysteroids and N-tert-butyl-N,N′-dibenzoylhydrazines, the binding activity toward the in vitro expressed EcR/USP complex using a wheat germ system was linearly correlated with that to the endogenous receptor proteins in the cell-free preparation of C. suppressalis integument. These results indicate that functional ecdysteroid receptor proteins were successfully expressed in a wheat germ protein synthesis system. © Pesticide Science Society of Japan.