A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins.
The silkworm Nd-s(D) mutant is silk fibroin-secretion deficient. In the mutant, a disulfide linkage between the heavy (H) and light (L) chains, which is essential for the intracellular transport and secretion of fibroin, is not formed because of a partial deletion of the L-chain gene. To utilize the inactivity of the mutant L-chain, we investigated the possibility of using the Nd-s(D) mutant for the efficient production of recombinant proteins in the silkworm. A germ line transformation of the mutant with a normal L-chain-GFP fusion gene was performed. In the transgenic mutant, normal development of the posterior silk gland (PSG) was restored and it formed a normal cocoon. The biochemical analysis showed that the transgenic silkworms expressed the introduced gene in PSG cells, produced a large amount of the recombinant protein, secreted it into the PSG lumen, and used it to construct the cocoon. The molar ratio of silk proteins, H-chain:L-chain-GFP:fibrohexamerin, in the lumen and cocoon in the transgenic silkworm was 6:6:1, and the final product of the fusion gene formed about 10% of the cocoon silk. This indicates that the transgenic mutant silkworm possesses the capacity to produce and secrete the recombinant proteins in a molar ratio equal to that of the fibroin H-chain, contributing around half molecules of the total PSG silk proteins.
Inoue
Satoshi
S
Insect Biotechnology and Sericology Department, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
Kanda
Toshio
T
Imamura
Morikazu
M
Quan
Guo-Xing
GX
Kojima
Katsura
K
Tanaka
Hiromitsu
H
Tomita
Masahiro
M
Hino
Rika
R
Yoshizato
Katsutoshi
K
Mizuno
Shigeki
S
Tamura
Toshiki
T
D000818:Animals; D012831:Bombyx / Q000235:genetics* / Q000378:metabolism*; D005349:Fibroins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D019476:Insect Proteins / Q000096:biosynthesis* / Q000235:genetics; D009154:Mutation; D030781:Organisms, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D019476:Insect Proteins; C432480:L-chain, fibroin protein, insect; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005349:Fibroins