Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

References

  1. Ramshaw, J.A.M., Werkmeister, J.A. & Glattauer, V. Collagen-based biomaterials. Biotechnol. Genet. Eng. Rev. 13, 335–382 (1996).

    CAS  Article  PubMed  Google Scholar 

  2. Sano, A., Hojo, T., Maeda, M. & Fujioka, K. Protein release from collagen matrices. Adv. Drug Deliv. Rev. 31, 247–266 (1998).

    CAS  Article  PubMed  Google Scholar 

  3. Cooperman, L. & Michaeli, D. The immunogenicity of injectable collagen. I. A 1-year prospective study. J. Am. Acad. Dermatol. 10, 638–646 (1984).

    CAS  Article  PubMed  Google Scholar 

  4. Tamura, T. et al. Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat. Biotechnol. 18, 81–84 (2000).

    CAS  Article  PubMed  Google Scholar 

  5. Lees, L.F. & Bulleid, N.J. The role of cysteine residues in the folding and association of the COOH-terminal propeptide of types I and III procollagen. J. Biol. Chem. 269, 24354–24360 (1994).

    CAS  PubMed  Google Scholar 

  6. Horn, C. & Wimmer, E.A. A versatile vector set for animal transgenesis. Dev. Genes Evol. 210, 630–637 (2000).

    CAS  Article  PubMed  Google Scholar 

  7. Rubin, G.M. & Spradling, A.C. Genetic transformation of Drosophila with transposable element vectors. Science 218, 348–353 (1982).

    CAS  Article  PubMed  Google Scholar 

  8. Handler, A.M. & Harrell II, R.A. Germline transformation of Drosophila melanogaster with the piggyBac transposon vector. Insect Mol. Biol. 8, 449–457 (1999).

    CAS  Article  PubMed  Google Scholar 

  9. Peloquin, J.J., Thibault, S.T., Staten, R. & Miller, T.A. Germ-line transformation of pink bollworm (Lepidoptera: Gelechiidae) mediated by the piggyBac transposable element. Insect Mol. Biol. 9, 323–333 (2000).

    CAS  Article  PubMed  Google Scholar 

  10. Nolan T., Bower, T.M., Brown, A.E., Crisanti, A. & Catteruccia, F. piggyBac-mediated germline transformation of the malaria mosquito Anopheles stephensi using the red fluorescent protein dsRED as a selectable marker. J. Biol. Chem. 277, 8759–8762 (2002).

    CAS  Article  PubMed  Google Scholar 

  11. Grzelak, K. Control of expression of silk protein genes. Comp. Biochem. Physiol. 110, 671–681 (1995).

    CAS  Article  Google Scholar 

  12. Bulleid, N.J., Wilson, R. & Lees, J.F. Type-III procollagen assembly in semi-intact cells: chain association, nucleation and triple-helix folding do not require formation of inter-chain disulphide bonds but triple-helix nucleation does require hydroxylation. Biochem. J. 317, 195–202 (1996).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Berg, R.A. & Prockop, D.J. The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen. Biochem. Biophys. Res. Commun. 52, 115–120 (1973).

    CAS  Article  PubMed  Google Scholar 

  14. Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T. & Kivirikko, K.I. Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase. EMBO J. 16, 6702–6712 (1997).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. John, D.C.A. et al. Expression of an engineered form of recombinant procollagen in mouse milk. Nat. Biotechnol. 17, 385–389 (1999).

    CAS  Article  PubMed  Google Scholar 

  16. Tomita, M. et al. Biosynthesis of recombinant human pro-α1(III) chains in a baculovirus expression system: production of disulphide-bonded and non-disulphide-bonded species containing full-length triple helices. Biochem. J. 312, 847–853 (1995).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Tomita, M., Kitajima, T. & Yoshizato, K. Formation of recombinant human procollagen I heterotrimers in a baculovirus expression system. J. Biochem. 121, 1061–1069 (1997).

    CAS  Article  PubMed  Google Scholar 

  18. Ruggiero, F. et al. Triple helix assembly and processing of human collagen produced in transgenic tobacco plants. FEBS Lett. 469, 132–136 (2000).

    CAS  Article  PubMed  Google Scholar 

  19. Shimura, K. Biochemical aspects on fibroin. Tanpakushitsu Kakusan Koso 24, 1324–1335 (1979).

    CAS  PubMed  Google Scholar 

  20. Kikuchi, Y., Mori, K., Suzuki, S., Yamaguchi, K. & Mizuno, S. Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain. Gene 110, 151–158 (1992).

    CAS  Article  PubMed  Google Scholar 

  21. Yamaguchi, K. et al. Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis. J. Mol. Biol. 210, 127–139 (1989).

    CAS  Article  PubMed  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Katsutoshi Yoshizato.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt771

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing