Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system.
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.
Kajikawa
Mizuho
M
Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Sasaki
Kaori
K
Wakimoto
Yoshitaro
Y
Toyooka
Masaru
M
Motohashi
Tomoko
T
Shimojima
Tsukasa
T
Takeda
Shigeki
S
Park
Enoch Y
EY
Maenaka
Katsumi
K
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
D043562:Receptors, G-Protein-Coupled; D011957:Receptors, Opioid; D011993:Recombinant Fusion Proteins; D004247:DNA; D000094942:Nociceptin Receptor