Transgenic silkworms produce recombinant human type III procollagen in cocoons

Masahiro Tomita; Hiroto Munetsuna; Tsutomu Sato; Takahiro Adachi; Rika Hino; Masahiro Hayashi; Katsuhiko Shimizu; Namiko Nakamura; Toshiki Tamura; Katsutoshi Yoshizato

doi:10.1038/nbt771

Transgenic silkworms produce recombinant human type III procollagen in cocoons

Nat Biotechnol. 2003 Jan;21(1):52-6. doi: 10.1038/nbt771. Epub 2002 Dec 16.

Authors

Masahiro Tomita¹, Hiroto Munetsuna, Tsutomu Sato, Takahiro Adachi, Rika Hino, Masahiro Hayashi, Katsuhiko Shimizu, Namiko Nakamura, Toshiki Tamura, Katsutoshi Yoshizato

Affiliation

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.

PMID: 12483223
DOI: 10.1038/nbt771

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.

Publication types

Evaluation Study
Validation Study

MeSH terms

Animals
Animals, Genetically Modified / genetics
Animals, Genetically Modified / metabolism
Bombyx / genetics*
Bombyx / metabolism*
Collagen Type III / biosynthesis*
Collagen Type III / genetics*
Collagen Type III / isolation & purification
Collagen Type III / therapeutic use
Drug Delivery Systems / methods
Feasibility Studies
Fibroins / biosynthesis
Fibroins / genetics
Gene Expression Regulation / genetics*
Green Fluorescent Proteins
Humans
Insect Proteins / biosynthesis
Insect Proteins / genetics
Luminescent Proteins / biosynthesis
Luminescent Proteins / genetics
Proteomics / methods
Pupa / metabolism
Recombinant Fusion Proteins / biosynthesis
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / isolation & purification
Tissue Engineering / instrumentation
Tissue Engineering / methods

Substances

Collagen Type III
Insect Proteins
L-chain, fibroin protein, insect
Luminescent Proteins
Recombinant Fusion Proteins
Green Fluorescent Proteins
Fibroins