Appl Microbiol Biotechnol (2010) 85:459–470 Table 1 Expression of recombinant proteins in silkworm larvae and pupae Proteins Intracellular protein Firefly luciferase Secretory proteins Human interferon-α Human macrophage colony-stimulating factor Human growth factor Rat interleukin-5 Human butyrylcholinesterase Bovine interleukin-21 Bovine interferon-t Porcine lactoferrin Human granulocyte macrophage colony- stimulating factor GFPuv-β3GnT2 fusion protein EGFP-spider dragline silk fusion protein Cholera toxin B Human stem cell factor anti-BSA scFV Human anti-BSA IgG1 Human α2,6-sialyltransferase Transmembrane proteins Human (pro)renin receptor Human prorenin-(pro)renin receptor complex Used viruses or bacmids Expression level BmNPV BmNPV BmNPV 13 mg per larva 50 mg in hemolymph 1 mg/10 larvae (after purification) BmNPV Cysteine protease depleted BmNPV BmNPV HyNPV Cysteine protease depleted BmNPV HyNPV BmNPV 160 μg/ml hemolymph (after purification) Kadono-Okuda et al. (1995) 1 mg/ml hemolymph 51 mg/4 larvae Ishihara et al. (1999) (after purification) 35 μg/ml hemolymph Wei et al. (2000) 50 μg/ml hemolymph Muneta et al. (2004) 4.6 mg/100 larvae (after purification) Nagaya et al. (2004) 20.5 mg/100 pupae (after purification) Wang et al. (2005) 100 μg per pupa Chen et al. (2006) References Palhan et al. (1995) Maeda et al. (1985) Qiu et al. (1994) 461 BmNPV bacmid BmNPV bacmid BmNPV BmNPV Cysteine protease and chitinase depleted BmNPV Cysteine protease and chitinase depleted BmNPV Cysteine protease and chitinase depleted BmNPV 91 μg/ml hemolymph 6 mg/a larva 54.4 μg/ml hemolymph 3 μg/ml hemolymph 188 μg/ml hemolymph 36 μg per larva Park et al. (2007) Zhang et al. (2008) Gong et al. (2005) Han et al. (2004) Ishikiriyama et al. (2009) Park et al. (2009) 2.2 mg/11 larvae (after purification) Ogata et al. (2009b) Cysteine protease depleted BmNPV Cysteine protease depleted BmNPV 31 μg per larva (after purification) 70 μg/15 larvae (after purification) Du et al. (2008) Du et al. (2009b) promoter. Efficient production of undegraded firefly lucif- erase was achieved (Suzuki et al. 1997). Using this BmNPV-CP− strain, 4.6 mg of bovine interferon-τ was purified from the hemolymph of 100 silkworm larvae (Nagaya et al. 2004). This expression level is at least tenfold higher than those of other expression systems. Moreover, insect-derived cellulose was expressed using BmNPV in which the cysteine protease and chitinase genes (BmNPV-CP−-Chi−) were deleted, to suppress larvae liquefaction caused by baculovirus infection (Lee et al. 2006). Several rounds of virus amplification steps are needed for BmNPV because the virus titer from B. mori cells is lower than that of Sf-9 cells, and it is more difficult to obtain a high titer of BmNPV than Autographa californica multiple nucleopolyhedrovirus (AcMNPV), which is generally used in the baculovirus expression system. To amplify BmNPV rapidly, a hybrid virus of AcMNPV and BmNPV was isolated and applied to the expression of recombinant proteins in silkworm larvae (Mori et al. 1992). A BmScH DNA fragment, the 572-bp SacI–HindIII fragment of the DNA helicase gene from BmNPV, determines the host specificity of BmNPV, and AcMNPV with the recombinant BmScH DNA fragment from BmNPV can infect BmN cells (Maeda et al. 1993). Two adjacent nucleotides (A and T) are the minimal essential sequence necessary to expand the host range of AcMNPV (Kamita and Maeda 1997). Muneta et al. (2004) reported that 500 μg of bovine interleukin-21 (IL-21) was purified from 30 ml of hemolymph using a hybrid baculovirus. Silkworm pupae have also been used as a bioreactor for recombinant protein production. Pupae can survive at 4°C for a long time and do not require mulberry leaves or an artificial diet during their growth. Human granulocyte macrophage colony-stimulating factor (hGM- CSF) was produced in pupae and purified from a homoge- nate (Chen et al. 2006). The hGM-CSF expressed in pupae