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. Deletion experiments showed the requirement of fibH(-5000/-3844) and fibH(-2211/-542) for the promoter activity. These two regions and fibH(-541/+24) that contained the basal promoter were tandem fused to yield fibH(-5000/-3844:-2211/-542:-541/+24), which was found to retain 88% of the activity of fibH(-5000/+24). Germline transgenic silkworms bearing fibH(-5000/-3844:-2211/-542:-541/+24) as a promoter and enhanced green fluorescent protein (EGFP) gene as a reporter efficiently secreted EGFP in cocoons. The promoter activity of fibH(-2211/-542) was further investigated, because this contained a DNase I-hypersensitive site. The transient expression assay demonstrated that the activity of fibH(-2211/-542) required fibH(-1659/-1590), which contained the homeodomain protein-binding motif. Mutation experiments suggested a critical role of the motif for the promoter activity. Electrophoretic mobility shift assay (EMSA) demonstrated that a nuclear protein of PSGs bound to the motif. We propose fibH(-1659/-1590) as a novel transcription enhancer that plays a key role for the expression by recruiting a homeodomain protein.

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.

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. Thus, it was concluded that the worms spun cocoons whose fibroin layers were composed of the inherent gene-derived natural fibroin (nF) and r(FL/bFGF). The mixture of nF and r(FL/bFGF) was dubbed r(FL/bFGF)nF. The solubilized r(FL/bFGF)nF was refolded using the glutathione redox system. Human umbilical vein endothelial cells (HUVECs) grew in the refolded r(FL/bFGF)nF-containing culture media, showing that bFGF in r(FL/bFGF) was biologically active. r(FL/bFGF)nF immobilized on a culture dish also supported the growth of HUVECs in bFGF-free media, suggesting the usefulness of r(FL/bFGF)nF as a new biomaterial for tissue engineering. The currently developed transgenic silkworms will be suitable for mass production of fibroins bearing a variety of biological activities.

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.

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 results showed that hr3 and IE1 cooperatively increased the ser1 promoter activity more than 30-fold. Then, transgenic silkworms were generated which expressed the EGFP with the signal peptide in MSG under the control of the hr3-linked ser1 promoter and IE1 gene. The silkworms exclusively secreted the EGFP into the sericin layer of cocoons as predicted. The expressed EGFP was extractable from cocoons through a simple procedure with neutral pH buffer solution. The expression system developed in this study enables us to produce recombinant proteins in bulk that can be easily extracted and purified.