Nature Biotechnology
21, 52 - 56 (2002)
Published online: 16 December 2002; | doi:10.1038/nbt771
Transgenic silkworms produce recombinant human type III procollagen in cocoonsMasahiro Tomita1, 4, Hiroto Munetsuna1, Tsutomu Sato1, 5, Takahiro Adachi1, 2, Rika Hino1, Masahiro Hayashi1, 2, Katsuhiko Shimizu1, Namiko Nakamura1, Toshiki Tamura3
& Katsutoshi Yoshizato1, 21
Hiroshima Tissue Regeneration Project, Hiroshima Prefecture Collaboration of Regional Entities for Advancement of Technological Excellence, Japan Science and Technology Corporation, 3-10-32, Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan
2
Developmental Biology Laboratory, Department of Biological Science, Graduate School of Science, Hiroshima University, 1-3-1, Kagamiyama, Higashihiroshima, Hiroshima 739-8526, Japan
3
National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan. 4
Permanent address: R&D Center, Terumo Corporation, 1500, Inokuchi, Nakai-machi, Ashigarakami-gun, Kanagawa 259-0151, Japan. 5
Permanent address: Koken Bioscience Institute, 1-13-10, Ukima, Kita-ku, Tokyo 115-0051, Japan.
Correspondence should be addressed to Katsutoshi Yoshizato kyoshiz@hiroshima-u.ac.jpWe describe the generation of transgenic silkworms that produce cocoons containing recombinant human collagen. A fusion cDNA was constructed encoding a protein that incorporated a human type III procollagen mini-chain with C-propeptide deleted, a fibroin light chain (L-chain), and an enhanced green fluorescent protein (EGFP). This cDNA was ligated downstream of the fibroin L-chain promoter and inserted into a piggyBac vector. Silkworm eggs were injected with the vectors, producing worms displaying EGFP fluorescence in their silk glands. The cocoons emitted EGFP fluorescence, indicating that the promoter and fibroin L-chain cDNAs directed the synthesized products to be secreted into cocoons. The presence of fusion proteins in cocoons was demonstrated by immunoblotting, collagenase-sensitivity tests, and amino acid sequencing. The fusion proteins from cocoons were purified to a single electrophoretic band. This study demonstrates the viability of transgenic silkworms as a tool for producing useful proteins in bulk.
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