462 had a molecular mass of 29 kDa, which is greater than that produced in the hemolymph of silkworm larvae. Moreover, the hGM-CSF expressed in pupae was glycosylated without its signal peptide. These results suggest that some comodi- fication and postmodifications are different between silk- worm larvae and pupae. BmNPV bacmid development and its applications To construct a recombinant baculovirus containing a gene of interest, cultured cells must be transfected with the baculovirus and the transfer vector. Moreover, this system requires the isolation and amplification of the recombinant baculovirus. It takes a long time (3–6 months) to prepare the high-titer recombinant baculovirus solution, and these steps are very tedious and troublesome. A novel system for the efficient production of recombinant AcMNPV was reported, which is based on site-specific transposition in Escherichia coli (Luckow et al. 1993). Luckow and colleagues constructed a recombinant baculovirus vector (bacmid) that can replicate in E. coli as a large plasmid. This system is known as the Bac-to-Bac baculovirus expression system, and various kits based on this Bac-to- Bac system have been distributed from Invitrogen Corp. This system reduces the time required to isolate and purify a recombinant baculovirus and allows the simultaneous construction of multiple recombinant baculoviruses. Motohashi et al. (2005) constructed a BmNPV bacmid and established the Bac-to-Bac system using BmNPV. GFPuv was expressed with only the injection of BmNPV bacmid DNA into silkworm larvae and pupae. This bacmid system provided rapid expression of recombinant proteins, since it did not require the preparation of a baculovirus solution by transfection, as compared to the baculovirus expression system using cultured cells (Fig. 1). Further- more, this BmNPV bacmid system dramatically reduced the time needed for recombinant protein production by silk- worm expression. Improved protein expression by a modified BmNPV bacmid Several proteins were produced using this BmNPV bacmid in silkworm larvae (Yue et al. 2006; Zhang et al. 2008). However, the degradation of recombinant proteins by baculoviral cysteine protease also occurred (Park et al. 2007). Therefore, the BmNPV-CP− bacmid was constructed, and the GFPuv-β1,3-N-acetylglucosaminyltransferase2 (β3GnT2) fusion protein (GGT2) was expressed successful- ly using this bacmid (Hiyoshi et al. 2007). The protease activity in the hemolymph of silkworm larvae injected with Appl Microbiol Biotechnol (2010) 85:459–470 the BmNPV-CP− bacmid was reduced by 85%, as compared to that in the hemolymph of BmNPV-bacmid-injected silkworm larvae. GGT2 degradation was also suppressed by using the BmNPV-CP− bacmid, and the β3GnT activity was improved by 30%. Moreover, the β3GnT activity of the hemolymph was improved 2.8-fold by using the BmNPV-CP−-Chi− bacmid over that obtained with the unmodified BmNPV bacmid (Park et al. 2008). Liquefac- tion of larvae was delayed by using the BmNPV-CP− and BmNPV-CP−-Chi− bacmids (Fig. 2). The expression level of human anti-bovine-serum-albumin (BSA) single-chain Fv (scFv) fragment in hemolymph was 3.8–4.3-fold higher with the BmNPV-CP− or BmNPV-CP−-Chi− bacmid than that with the unmodified BmNPV because of the suppres- sion of degradation by the cysteine protease from BmNPV (Fig. 2). However, no difference in the scFv expression levels between the modified and unmodified bacmids was observed in pupae (Ishikiriyama et al. 2009). This result suggests that the baculoviral cysteine protease, v-cath, may not be activated or only a small portion of the cysteine protease may be expressed in pupae. Pupae may be useful for the production of cysteine protease-sensitive proteins. A hybrid nucleopolyhedrovirus (HyNPV) bacmid was also constructed, by cotransfection with the AcMNPV bacmid and pUC18/BmScH containing the BmScH frag- ment of the DNA helicase gene from BmNPV (Deo et al. 2006). This bacmid allows baculoviral amplification in AcMNPV and protein expression in BmNPV. To obtain a high virus titer, this HyNPV bacmid is very helpful. Molecular chaperones play an important role in protein folding at the endoplasmic reticulum. Bacmids allow easy coexpression of a chaperone with a target protein. The expression level of a recombinant GFPuv-human α1,4-N- acetylglucosaminyltransferase (α4GnT) fusion protein was enhanced in silkworm larvae, by coexpression of a human molecular chaperone, human calnexin (CNX), or immuno- globulin heavy-chain binding protein (BiP; Nakajima et al. 2009). The expression of CNX and BiP under the control of the polyhedrin promoter enhanced the α4GnT activity by 1.4- and 2.0-fold, respectively. When CNX or BiP under the control of the ie-2 promoter was expressed during the early stage of baculovirus infection, the α4GnT activity in the hemolymph was also enhanced significantly. In silkworm larvae, the coexpression of a chaperone with the target protein effectively increases the quality of the recombinant protein. Protein production using transgenic silkworms Transgenic silkworms have also been used for recombinant protein production. To establish transgenic silkworms, two different systems, involving the use of an attenuated recombinant baculovirus or a piggyBac transposon-derived