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Transgenic silkworms produce recombinant human type III procollagen in cocoons

Nature Biotechnology volume 21, pages 52–56 (2003)Cite this article

Abstract

We 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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Figure 1: Structures of fusion cDNAs of LE, MOSRA-7, and MOSRA-8 and of the vectors pLE, pMOSRA-7, and pMOSRA-8.
Figure 2: Fluorescence of DsRed in transgenic silkworms bearing pMOSRA-7.
Figure 4: The fluorescence of EGFP in pMOSRA-7-bearing transgenic silkworms and their cocoons.
Figure 3: Genomic Southern blot hybridization.
Figure 5: Analysis of cocoon proteins.

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Acknowledgements

We thank Dr. Ernst A. Wimmer of Universität Bayreuth for kindly providing us with pBac[3xP3-EGFPafm] and Dr. Shigeki Mizuno at Nihon University and Dr. Satoshi Inoue at the National Institute of Agrobiological Sciences for kindly providing anti-fibroin L-chain antibodies.

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Authors and Affiliations

  1. 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

    Masahiro Tomita, Hiroto Munetsuna, Tsutomu Sato, Takahiro Adachi, Rika Hino, Masahiro Hayashi, Katsuhiko Shimizu, Namiko Nakamura & Katsutoshi Yoshizato

  2. Department of Biological Science, Developmental Biology Laboratory, Graduate School of Science, Hiroshima University, 1-3-1, Kagamiyama, Higashihiroshima, Hiroshima, 739-8526, Japan

    Takahiro Adachi, Masahiro Hayashi & Katsutoshi Yoshizato

  3. National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8634, Japan

    Toshiki Tamura

  4. R&D Center, Terumo Corporation, 1500, Inokuchi, Nakai-machi, Ashigarakami-gun, Kanagawa, 259-0151, Japan

    Masahiro Tomita

  5. Koken Bioscience Institute, 1-13-10, Ukima, Kita-ku, Tokyo, 115-0051, Japan

    Tsutomu Sato

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  1. Masahiro Tomita
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Correspondence to Katsutoshi Yoshizato.

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Tomita, M., Munetsuna, H., Sato, T. et al. Transgenic silkworms produce recombinant human type III procollagen in cocoons. Nat Biotechnol 21, 52–56 (2003). https://doi.org/10.1038/nbt771

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