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Transgenic silkworms produce recombinant human type III procollagen in cocoons

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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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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