title: A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins.
abstract: 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.
authors.affiliation: Insect Biotechnology and Sericology Department, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Inoue
authors.forename: Toshio
authors.initials: T
authors.lastname: Kanda
authors.forename: Morikazu
authors.initials: M
authors.lastname: Imamura
authors.forename: Guo-Xing
authors.initials: GX
authors.lastname: Quan
authors.forename: Katsura
authors.initials: K
authors.lastname: Kojima
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Tanaka
authors.forename: Masahiro
authors.initials: M
authors.lastname: Tomita
authors.forename: Rika
authors.initials: R
authors.lastname: Hino
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Yoshizato
authors.forename: Shigeki
authors.initials: S
authors.lastname: Mizuno
authors.forename: Toshiki
authors.initials: T
authors.lastname: Tamura
mesh_terms: 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*
chemical_list: D019476:Insect Proteins; C432480:L-chain, fibroin protein, insect; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005349:Fibroins