Appl Microbiol Biotechnol (2010) 85:459–470 (A) AcMNPV bacmid system Within a week DH10Bac Destination vector Bacmid construction Bacmid extraction Transfection 2-3 weeks Virus amplification (Sf-9 cells) Virus infection (insect cells) 463 4-5 weeks 3-4 days Recovery of protein from supernatant or cells PCR fragment (B) BmNPV expression system (conventional system) 3-6 months 12-25 weeks 4-5 days Recovery of protein from hemolymph or fat body Transfer vector Co-transfection Virus Virus purification Amplification (Bm cells) Infection of virus (C) BmNPV bacmid system Within a week DH10BmBac Destination vector Bacmid construction Bacmid extraction Injection of bacmid 2-3 weeks 4-6 days Recovery of protein from hemolymph or fat body Fig. 1 Baculovirus gene expression system in insect cells and silkworm larvae. a AcMNPV expression system using the AcMNPV bacmid. The target gene was incorporated into the AcMNPV bacmid. This extracted recombinant AcMNPV bacmid from E. coli was transfected into insect cells. The resulting recombinant AcMNPV was purified, amplified, and increased in titer and then was used for insect cell infection. b Conventional BmNPV expression system. The target gene and the wild-type BmNPV gene were cotransfected into B. mori cells, and the recombinant BmNPV was obtained. This recombinant BmNPV bacmid from E. coli was injected directly into silkworm larvae. After 4–6 days postinjection, the recombinant protein was harvested vector, were adopted (Tamura et al. 2000; Yamao et al. 1999). A method combining the two systems was also established (Yamamoto et al. 2004). Human type III procollagen and feline interferon were produced in cocoons using transgenic silkworms (Kurihara et al. 2007; Tomita et al. 2003). Human μ-opioid receptor was expressed in the silk glands and fat bodies of transgenic silkworms, which were screened by the GAL4/UAS system (Tateno et al. 2009). Its expression level was comparable to that obtained in the baculovirus expression system using Sf-9 cells. N-glycan structure produced by silkworm larvae Insect cells can assemble N-glycan precursors to produce high mannose or paucimannosidic end products. However, the cells failed to elongate the trimmed N-glycan to produce complex products containing terminal galactose and sialic acid (SA) residues (Kost et al. 2005). Enzyme assays revealed that the insect cell lines had little or none of the galactosyltransferase and sialyltransferase activities in- volved in N-glycan elongation. When human anti-BSA IgG1 was expressed in hemo- lymph, using the BmNPV-CP−-Chi− bacmid, its main N- glycan structure was paucimannosidic (Park et al. 2009). In accordance with this observation, human killer cell Ig-like receptor 2DL1 was expressed using the BmNPV bacmid (see details below), and the protein thus obtained harbored the paucimannose-type oligosaccharides, Manalpha1- 6Manbeta1-4GlcNAcbeta1-4GlcNAc and Manalpha1- 6Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc (Sasaki et al. 2009). However, the N-glycan of β3GnT2 purified from the hemolymph of larvae contained terminal N- acetylglucosamine (GlcNAcβ1,2Manα1,3(GlcNAcβ1,4) (Manα1,6)Manβ1,4GlcNAcβ1,4GlcNA) and galactose residues (Galβ1,4GlcNAcβ1,2Manα1,3(GlcNAcβ1,4) (Manα1,6)Manβ1,4GlcNAcβ1,4GlcNAc), which were not observed in samples from cultured cells (Dojima et al. 2009). These results indicate that β1,2N-acetylglucosami- nyltransferase I (GlcNAcT I), β1,4N-galactosyltransferase (GalT), and β1,4N-acetylglucosaminyltransferase III (GlcNAcT III) might exist in silkworm larvae, but not in insect cells (Fig. 3). Therefore, silkworm larvae would be useful for the production of human glycoproteins, if the