460 cells but also insect larvae and pupae can be used for protein production. The silkworm, Bombyx mori, has been used for silk production for centuries and recently also for protein production, as a bioreactor. Since Maeda et al. (1985) reported the production of human α-interferon in silkworm larvae, using recombinant B. mori nucleopolyhedrovirus (BmNPV), the production of many proteins has been achieved. TORAY Ind. Inc. (Tokyo, Japan) used silkworms to produce two recombinant proteins for veterinary use. Intercat, a drug principally composed of feline interferon, for feline calicivirus infections (commonly known as “cat flu”) was introduced in February 1994 as the world’s first antiviral drug for animal use. Interdog, a drug principally composed of canine interferon-γ for canine atopic derma- titis, was launched in December 2005. Nowadays, recom- binant proteins produced from silkworms are being investigated for commercial purposes. This review provides an overview of the production of recombinant proteins using silkworm larvae and pupae and the improvement of silkworm expression systems by the development of the BmNPV bacmid. Silkworm genome sequence Silkworms are the larvae of the silk moth, B. mori. B. mori was domesticated from its wild ancestor, Bombyx mandar- ina, and has been used for silk production for about 5,000 years. Silkworm silk in the natural form consists of silk fibroin, the core filaments, and silk sericin, a coating protein surrounding fibroin. Sericin is removed in commer- cial silk, and this silk has been used in textile production for centuries. B. mori is an important model organism of Lepidoptera in the fields of genetics, physiology, and biochemistry. Two whole-genome shotgun (WGS) sequencing projects for B. mori were reported in 2004 (Mita et al. 2004; Xia et al. 2004), and its expressed sequence tag database was constructed (Mita et al. 2003). In 2008, two data sets from WGS projects were merged and assembled with new data (International Silkworm Genome Consortium 2008). The silkworm has 28 chromosomes and an estimated genome size of around 500 Mb. A large number of transposable elements (TEs) exist in the B. mori genome, accounting for 43.5% of the whole. TEs occupy from 2.7% to 25% in the whole genome in 12 Drosophila species and 47% in Aedes aegypti (Mita et al. 2004). The gene-finder algorithm BGF (BGI GeneFinder) predicted that approximately 18,510 genes are encoded in the silkworm genome. This gene number in the silkworm genome is higher than that in Drosophila melanogaster (13,379 genes), which belongs to Diptera (Drosophila 12 Genome Consortium 2007; Nene Appl Microbiol Biotechnol (2010) 85:459–470 et al. 2007; International Silkworm Genome Consortium 2008). Moreover, orthology relationship of genes from B. mori, D. melanogaster, A. aegypti, and Caenorhabditis elegans by phylogenetic methods showed 13,450 gene families. Approximately 11.45% and 25.9% of the gene families are specific to insects and vertebrates, respectively, and 3,223 genes are silkworm specific (The International Silkworm Genome Consortium 2008). A genome-wide microarray analysis of fifth-instar larvae of the silkworm identified tissue- and developmental-stage-specific genes (Xia et al. 2007). Tissue-specific genes and genes differen- tially expressed in different tissues were identified. More- over, to construct the secondary protein database of the silkworm, proteomic identification of protein-coding genes was performed by tandem mass spectrometry, using silkworm pupae as the sample (Zhang et al. 2009). Baculovirus expression system in silkworm larvae or pupae Silkworm larvae have been used as a bioreactor for recombinant protein production for decades. Maeda et al. (1985) first reported the production of human interferon alpha (IFN-α) in the hemolymph of silkworm larvae using BmNPV, containing the gene encoding human α-interferon driven by the polyhedrin promoter. Recombinant proteins expressed in silkworm larvae are shown in Table 1. Many proteins from eukaryotes were expressed in silkworm larvae and purified. In general, the expression level of recombinant proteins in silkworm larvae is higher than that in cultures cells from insect and animals. The mouse interleukin-3 activity in the hemolymph of silkworm larvae was about 20-fold and 10,000-fold higher than those in the culture supernatants of BmN cells and COS7 cells, respectively (Miyajima et al. 1987). The hemolymph of infected silkworms showed human butyrylcholinesterase activities 23- and 280-fold higher than those in BmN cells and Chinese hamster ovary (CHO) cells, respectively (Wei et al. 2000). One milligram of purified human macrophage colony-stimulating factor (CSF) was obtained from the hemolymph of ten silkworm larvae, and 160 μg of human growth factor was purified from 1 ml of hemolymph (Kadono-Okuda et al. 1995; Qiu et al. 1994). The degradation of the expressed proteins is often observed in silkworm larvae. This is probably caused by a baculovirus cysteine protease, v-cath, in the baculovirus- insect cell expression system (Kadono-Okuda et al. 1995). To overcome this problem, BmNPV lacking the cysteine protease gene (BmNPV-CP−) was constructed by cotrans- fection with BmNPV genome DNA and pBmFCPdLZ containing the cysteine protease gene partially substituted with the β-galactosidase gene fused with the hsp70