Transgenic silkworms produce recombinant human type III procollagen in cocoons.
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.
Tomita
Masahiro
M
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.
Munetsuna
Hiroto
H
Sato
Tsutomu
T
Adachi
Takahiro
T
Hino
Rika
R
Hayashi
Masahiro
M
Shimizu
Katsuhiko
K
Nakamura
Namiko
N
Tamura
Toshiki
T
Yoshizato
Katsutoshi
K
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism; D012831:Bombyx / Q000235:genetics* / Q000378:metabolism*; D024061:Collagen Type III / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000627:therapeutic use; D016503:Drug Delivery Systems / Q000379:methods; D005240:Feasibility Studies; D005349:Fibroins / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D019476:Insect Proteins / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D040901:Proteomics / Q000379:methods; D011679:Pupa / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D023822:Tissue Engineering / Q000295:instrumentation / Q000379:methods
D024061:Collagen Type III; D019476:Insect Proteins; C432480:L-chain, fibroin protein, insect; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005349:Fibroins