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 (K D =2.1 nM). For a series of ecdysteroids and N-tert-butyl-N,NЈ-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.

New reporter gene assays for detecting natural and synthetic molting hormone agonists using yeasts expressing ecdysone receptors of various insects

“…The amplified fragments were digested with Sal I and Hin dIII for DmEcR, and Sma I and Eco RI for DmUSP, and cloned into the corresponding sites of multicloning site (MCS) 1 and MCS2 of the expression vector pUdp6 , respectively. Similarly, CsEcRB1 () and CsUSP () were amplified from the plasmids pEU3‐NII‐EcR and pEU3‐NII‐USP, respectively . LdEcRA () and LdUSP () ORFs were derived from pET‐23a(+)‐LdEcR and pET‐23a(+)‐USP , respectively.…”

Cell-Based Screening Systems for Insecticides

“…More recently, after the cloning of complete ORFs of EcR and USP, in vitro transcription/translation systems were used to evaluate binding af fi nity and speci fi city for a particular species (Minakuchi et al 2003(Minakuchi et al , 2004(Minakuchi et al , 2007Ogura et al 2005a ;review by Nakagawa 2005 ;Nakagawa and Henrich 2009 ) . Other assays employed organ cultures to evaluate molting hormone activity such as those based on the evagination of imaginal discs (Smagghe et al 2000 ) and the promotion of chitin synthesis by cuticle fragments (Nakagawa et al 1998 ;review by Nakagawa 2005 ) .…”

Structureactivity relationship and mode of action study of insect growth regulators

“…Chilo suppressalis 15,16 Spodoptera exigua 17,18 Leptinotarsa decemlineata 19,20 piperonyl butoxide PB LD 50 , mmol/insect pLD 50 Table 3 C. suppressalis Table 3 49 Not determined. …”