TY - JOUR
T1 - Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system
AU - Kajikawa, Mizuho
AU - Sasaki, Kaori
AU - Wakimoto, Yoshitaro
AU - Toyooka, Masaru
AU - Motohashi, Tomoko
AU - Shimojima, Tsukasa
AU - Takeda, Shigeki
AU - Park, Enoch Y.
AU - Maenaka, Katsumi
N1 - Funding Information:
We thank M. Sasaki, M. Ohtsu, and D. Kohda (Medical Institute of Bioregulation) and stuff of the Research Support Center, Graduate School of Medical Sciences, Kyushu University. K.M. was supported in part by the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, and the Japan Bio-oriented Technology Research Advancement Institute (BRAIN).
PY - 2009/7/31
Y1 - 2009/7/31
N2 - 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 (Giα) 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]GTPγS-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.
AB - 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 (Giα) 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]GTPγS-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.
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U2 - 10.1016/j.bbrc.2009.05.063
DO - 10.1016/j.bbrc.2009.05.063
M3 - Article
C2 - 19463790
AN - SCOPUS:67349205441
VL - 385
SP - 375
EP - 379
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 3
ER -