Structure and function of 5'-flanking regions of Bombyx mori fibroin heavy chain gene: identification of a novel transcription enhancing element with a homeodomain protein-binding motif.

We studied the promoter activity of a 5'-flanking region from -5000 to +24 (-5000/+24) in Bombyx mori fibroin heavy chain gene (fibH), fibH(-5000/+24). A luciferase reporter vector carrying fibH(-5000/+24) was bombarded to isolated posterior silk glands (PSGs). The PSGs showed a high luciferase activity when transplanted to larvae, indicating its potent promoter activity.

A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon.

A silk thread of the silkworm, Bombyx mori, is composed of the insoluble inner fibroin and the hydrophilic outer sericin layer, which are synthesized in the posterior and middle silk gland (MSG), respectively. This study aimed to develop a novel sericin 1 gene (ser1) promoter-driven recombinant expression system using transgenic silkworms, in which recombinant proteins are synthesized in MSG and secreted into the sericin layer. To obtain a high level of gene expression, we tested whether a baculovirus-derived enhancer, hr3, and a trans-regulator, IE1, are capable of stimulating the transcriptional activity of the ser1 promoter, using a transient gene expression system.

The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor.

We generated germline transgenic silkworms bearing a fibroin light chain (FL) promoter-driven FL gene whose 3'-end was flanked with human basic fibroblast growth factor (bFGF) gene, FL/bFGF gene. The cocoons from transgenic worms were trypsinized to remove sericin layers, and treated with solution containing CaCl(2), ethanol, and water at a molar ratio of 1:2:8 (CaCl(2)/ethanol/water) to solubilize fibroin layers. Western blot analysis showed that the recombinant protein, r(FL/bFGF), was solubilized with CaCl(2)/ethanol/water, but not with trypsin, indicating that r(FL/bFGF) was in fibroin layers.

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.

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.