title: Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system.
abstract: Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G(i)alpha) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [35S]GTPgammaS-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.
authors.affiliation: Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Mizuho
authors.initials: M
authors.lastname: Kajikawa
authors.forename: Kaori
authors.initials: K
authors.lastname: Sasaki
authors.forename: Yoshitaro
authors.initials: Y
authors.lastname: Wakimoto
authors.forename: Masaru
authors.initials: M
authors.lastname: Toyooka
authors.forename: Tomoko
authors.initials: T
authors.lastname: Motohashi
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Shimojima
authors.forename: Shigeki
authors.initials: S
authors.lastname: Takeda
authors.forename: Enoch Y
authors.initials: EY
authors.lastname: Park
authors.forename: Katsumi
authors.initials: K
authors.lastname: Maenaka
mesh_terms: D000818:Animals; D012831:Bombyx / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D017924:Nucleopolyhedroviruses / Q000235:genetics; D014176:Protein Biosynthesis; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000235:genetics; D011957:Receptors, Opioid / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D000094942:Nociceptin Receptor
chemical_list: D043562:Receptors, G-Protein-Coupled; D011957:Receptors, Opioid; D011993:Recombinant Fusion Proteins; D004247:DNA; D000094942:Nociceptin Receptor