Appl Microbiol Biotechnol (2010) 85:459–470 The sugar modifications of the obtained KIR2DL1 protein revealed that the protein harbored the relatively small paucimannose-type oligosaccharides and thus seemed to be appropriate for structural and binding studies of cell surface receptors. GPCRs are seven-transmembrane proteins and thus are quite difficult to express due to their low solubility and instability. The expression of a GPCR (human μ-opioid receptor) in silkworms was first reported using a transgenic technique, but the expression level was rather low, and many technical steps were required (Tateno et al. 2009). The expression of other GPCRs using other expression systems including Sf9 and CHO cells was reviewed by Massotte (2003) and Sarramegna et al. (2003). Human GPCR (human nociceptin receptor) expression was reported recently by the simple injection of BmNPV bacmid DNA into the silkworm (Kajikawa et al. 2009). Human nociceptin receptor, which is an inhibitory GPCR for an exogenous opioid peptide, nociceptin, involved in pain control ( Meunier 1997), was actually produced as a membrane protein in microsomal fractions of the fat bodies of silkworms. The microsomal fractions including func- tional GPCRs could be very easily prepared by a simple operation and centrifugation. Interestingly, the protein was also expressed in the BmNPV viral fraction, which was consistent with that previously reported for the GPCR expression in Sf9 cells (Masuda et al. 2003). GPCRs physiologically bind at the cytoplasmic region to a trimeric G protein, composed of alpha (Gα), beta (Gβ), and gamma (Gγ) subunits (Wess 1997). The ligand binding to GPCRs enhances the guanosine triphosphatase (GTPase) activity of the trimeric G protein, resulting in its dissociation from GPCRs by exchanging Gα-bound GTP to GDP, which triggers the G-protein-mediated signal transduction (Liebmann and Bohmer 2000). The nociceptin receptor fused with the G protein alpha subunit (Giα), whose ligand recognition can be detected by measuring the [35S]GTPγS binding, was also successfully expressed in the same system as the nonfused protein as described above. This expression system exhibited sufficient production for 500 binding assays using the radiolabeled compound, [35 S]GTPγS. The expression level of the nonfusion type of nociceptin receptor was much higher than that of the fusion type. Silkworms also have the capacity to produce functional membrane proteins in the same way as secretory proteins. Many membrane proteins have been produced in the hemolymph and pupae of silkworms as secretory proteins. Recently, human (pro)renin receptor (hPRR) and its complex with human prorenin were expressed in silkworm larvae, with the same expression level as some secretory proteins, and were purified from the fat bodies of larvae (Du et al. 2008, 2009a). This hPRR may be helpful for the development of an hPRR blocker. 467 Conclusions and future developments The BmNPV bacmid system is innovative and easy to use for producing large amount of recombinant protein, without any laborious techniques. Many BmNPV DNAs for recombinant protein expression can be constructed rapidly by the BmNPV bacmid system, based on the Bac-to-Bac system. Moreover, expression of multi- subunit complexes can be achieved simply by injection of a mixture of BmNPV bacmids into silkworms, without prior virus amplification using cultured cells. The protein expression levels in bacmid-injected silkworm larvae are very high, which reduces the cost of large-scale produc- tion. This BmNPV bacmid-silkworm expression strategy is very successful for expression of membrane proteins including GPCRs which are major drug targets. There- fore, this system is very promising and will be useful for high-throughput drug screening of many transmembrane proteins and orphan GPCRs for which physiological ligands have not yet been identified (Takeda et al. 2002; Wise et al. 2004). In Asian countries, silkworms are abundantly available, and many laboratories have experience in rearing and maintaining larvae. Thus, the opportunity to utilize the long-forgotten resourceful silkworm for producing thera- peutically important proteins, vaccines, and biomaterials is here. By the spurt in influenza viruses, vaccines for influenza viruses are being actively produced in insect cell system. More and more patents are being procured for customized insect cell lines and their products are making inroads into the market which until now had been dominated by other production systems. With the genomic elucidation of silkworm, even more laboratories around the world will be interested in its utilization. We expect that the glory of silkworm is returning back, and it will be once more associated with wealth and prosperity. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. References Boublik Y, Bonito PD, Jones IM (1995) Eukaryotic virus display: engineering the major surface glycoprotein of the Autographa californica nuclear polyhedrosis virus (AcNPV) for the presen- tation of foreign proteins on the virus surface. Bio/Technol 13:1079–1084 Chen J, Wu XF, Zhang YZ (2006) Expression, purification and characterization of human GM-CSF using silkworm pupae (Bombyx mori) as a bioreactor. J Biotechnol 123:236–247