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
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Ramshaw, J.A.M., Werkmeister, J.A. & Glattauer, V. Collagen-based biomaterials. Biotechnol. Genet. Eng. Rev. 13, 335–382 (1996).
Sano, A., Hojo, T., Maeda, M. & Fujioka, K. Protein release from collagen matrices. Adv. Drug Deliv. Rev. 31, 247–266 (1998).
Cooperman, L. & Michaeli, D. The immunogenicity of injectable collagen. I. A 1-year prospective study. J. Am. Acad. Dermatol. 10, 638–646 (1984).
Tamura, T. et al. Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat. Biotechnol. 18, 81–84 (2000).
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).
Horn, C. & Wimmer, E.A. A versatile vector set for animal transgenesis. Dev. Genes Evol. 210, 630–637 (2000).
Rubin, G.M. & Spradling, A.C. Genetic transformation of Drosophila with transposable element vectors. Science 218, 348–353 (1982).
Handler, A.M. & Harrell II, R.A. Germline transformation of Drosophila melanogaster with the piggyBac transposon vector. Insect Mol. Biol. 8, 449–457 (1999).
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).
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).
Grzelak, K. Control of expression of silk protein genes. Comp. Biochem. Physiol. 110, 671–681 (1995).
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).
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).
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).
John, D.C.A. et al. Expression of an engineered form of recombinant procollagen in mouse milk. Nat. Biotechnol. 17, 385–389 (1999).
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).
Tomita, M., Kitajima, T. & Yoshizato, K. Formation of recombinant human procollagen I heterotrimers in a baculovirus expression system. J. Biochem. 121, 1061–1069 (1997).
Ruggiero, F. et al. Triple helix assembly and processing of human collagen produced in transgenic tobacco plants. FEBS Lett. 469, 132–136 (2000).
Shimura, K. Biochemical aspects on fibroin. Tanpakushitsu Kakusan Koso 24, 1324–1335 (1979).
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).
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).
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.
The authors declare no competing financial interests.
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
The Nucleus (2022)
Nano-Micro Letters (2021)
In vitro assessment of antitumor immune responses using tumor antigen proteins produced by transgenic silkworms
Journal of Materials Science: Materials in Medicine (2021)
Transdermal peptide conjugated to human connective tissue growth factor with enhanced cell proliferation and hyaluronic acid synthesis activities produced by a silkworm silk gland bioreactor
Applied Microbiology and Biotechnology (2020)
Transgenic Research (2020)