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

Tomita, Masahiro; Hino, Rika; Ogawa, Shingo; Iizuka, Masaaki; Adachi, Takahiro; Shimizu, Katsuhiko; Sotoshiro, Hisaya; Yoshizato, Katsutoshi

doi:10.1007/s11248-007-9087-x

Cited by 74 publications

(50 citation statements)

References 32 publications

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“…The primary and secondary cleavage sites of the SerUAS-FHX_int-EGFP protein were Ala17/Gly18 and Gly18/Leu19, respectively. These results indicate that the signal peptide-cleaving activity originally described in MSG cells of non-transgenic silkworms (Liu et al 2006) is retained in transgenic silkworms in our binary Ser-GAL4/modified UAS system and that mammalian signal peptides are also recognized in this system, as previously reported (Adachi et al 2006;Tomita et al 2007). …”

Section: Expression Of Egfp and Gal4 Genes In Msgs Of A Ser1-gal4/uassupporting

confidence: 86%

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Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori

et al. 2009

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To construct an efficient system for the production of recombinant proteins in silkworm (Bombyx mori), we investigated the promoter activity of the silkworm sericin 1, 2, and 3 genes (Ser1, Ser2, and Ser3) using a GAL4/UAS binary gene expression system in transgenic silkworm. The promoter activity of the upstream region of Ser1 was strong, yielding high expression of an enhanced green fluorescent protein (EGFP) transgene in the middle and posterior regions of the middle silk gland (MSG) after day 2 of the fifth instar. The Ser3 upstream region exhibited moderate promoter activity in the anterior MSG, but the Ser2 upstream region did not exhibit any promoter activity. Since the strongest promoter activity was observed for Ser1, we devised a system for the production of recombinant proteins using a GAL4-Ser1 promoter construct (Ser1-GAL4). Transgenic silkworms harboring both the Ser1-GAL4 construct and the previously reported upstream activating sequence (UAS)-EGFP construct, which contains the TATA box region of the Drosophila hsp70 gene, yielded approximately 100 microg EGFP per larva. When we then analyzed the TATA box region, signal peptide, and intron sequences for their effects on production from the UAS-EGFP construct, we found that the optimization of these sequences effectively increased production to an average of 500 microg EGFP protein per transgenic larva. We conclude that this binary system is a useful tool for the mass production of recombinant proteins of biomedical and pharmaceutical interest in silkworm.

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Section: Expression Of Egfp and Gal4 Genes In Msgs Of A Ser1-gal4/uassupporting

confidence: 86%

“…Efforts to facilitate the extraction of recombinant protein have led to the development of a Ser1 promoter-driven expression system that uses a baculovirus-derived enhancer (hr3) and transregulator (IE1; Ogawa et al 2007;Tomita et al 2007). This system efficiently produced recombinant human serum albumin and EGFP in MSGs.…”

Section: Introductionmentioning

confidence: 99%

Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori

et al. 2009

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“…In the MSG-expression system, reported by Tomita et al (2007), the BmNPV-derived enhancer, hr3, and trans-activator, IE1, were used to stimulate the ser1 promoter. The use of hr3 and IE1 cooperatively enhanced the ser1 promoter activity more than 30-fold ).…”

Section: Expression Of Recombinant Proteins In the Msgmentioning

confidence: 99%

Transgenic silkworms that weave recombinant proteins into silk cocoons

Tomita

2010

Biotechnol Lett

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As a result of breeding for more than 4,000 years, the silkworm, Bombyx mori, has acquired the ability to synthesize bulk amounts of silk proteins in its silk glands. To utilize this capacity for mass production of useful proteins, transgenic silkworms were generated that synthesized recombinant proteins in the silk gland and secreted them into the silk cocoon. The silk gland is classified into two main regions: the posterior (PSG) and the middle silk gland (MSG). By controlling the expressed regions of the recombinant protein gene in the silk gland, we were able to control the localization of the synthesized protein in the silk thread. Expression in the PSG or MSG led to localization in the insoluble fibroin core or hydrophilic outer sericin layer, respectively. This review focuses on the expression of recombinant protein in the MSG of transgenic silkworms. The recombinant protein secreted in the sericin layer is extractable from the cocoon with only a small amount of endogenous silk protein contamination by soaking the cocoon in mild aqueous solutions. The possibility of utilizing transgenic silkworms as a valuable tool for the mass production of therapeutic and industrially relevant recombinant proteins is discussed.

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“…7,8) Furthermore, the modification of silk protein properties by transgenic silkworms is now being attempted. Takabayashi reported the applicability of silk fibroin, expressed with a cell attachment factor, in the construction of biocompatible and biodegradable artificial blood vessels.…”

Section: )mentioning

confidence: 99%

“…1) However, recent progress in transgenic technology has produced strains of transgenic silkworms that secrete recombinant proteins such as vaccines and cytokines in the SC of their cocoons. 7,8) Furthermore, the modification of silk protein properties by transgenic silkworms is now being attempted. Takabayashi reported the applicability of silk fibroin, expressed with a cell attachment factor, in the construction of biocompatible and biodegradable artificial blood vessels.…”

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Use of Silk Protein, Sericin, as a Sustained-Release Material in the Form of a Gel, Sponge and Film

et al. 2010

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Sericin (SC) is a component of silk protein in the silkworm cocoon. Silk protein consists of fibroin as the fiber and SC as the glue. SC comprises 25-30% of the silkworm (Bombyx mori) cocoon.1) Although SC is composed of 18 different amino acids, it contains a high number of polar side chains with hydroxyl, carboxyl and amino groups.1) Isolation and characterization of SC components from the cocoon of Bombyx mori showed that SC primarily consists of three polypeptides with molecular weights of 150, 250 and 400 kDa. The phenylthiocarbamyl (PTC) method 2) showed that the amino acid compositions of all three SC have high contents of serine (Ser, 33.2-39.0%), glycine (Gly, 14.1-16.0%) and aspartic acid/asparagine (Asp/Asn, 11.3-15.7%). The structural analysis and cloning of SC genes Ser1 and Ser2 (Src-2) have been described. [3][4][5][6] Correspondence of the amino acid composition of SC with these genes suggested that the 150-and 400-kDa SCs correspond to Ser1 proteins (77-331 kDa) encoded by the Ser1 gene, and that the 250-kDa SC corresponds to S-2 protein (227 kDa) encoded by the Src-2 gene.2) The most abundant component is the largest SC (400 kDa), which corresponds to the Ser1C protein (331 kDa).2) A repetitive 38-amino acid sequence rich in Ser (40%) dominates a large part of the Ser1C protein and is predicted to have a strong tendency to form b-sheet structure. Another part of the Ser1C protein is hydrophilic and has a high content of charged residues including acidic (glutamic acid (Glu) and Asp) and basic (lysine (Lys) and arginine (Arg)) amino acids. 6) In contrast, the 250-kDa SC polypeptide, which corresponds to S-2 protein (227 kDa), has less bsheet forming propensity and higher hydrophilicity than the 150-and 400-kDa polypeptides. This is because the 250-kDa SC contains larger amounts of b-sheet breaking residues like Glu, glutamine (Gln) and Lys, and smaller amounts of bsheet favoring residues like threonine (Thr) and tyrosine (Tyr) compared to the 150-and 400-kDa SCs. 2)Historically, sericulture was widely carried out to support the silk spinning and weaving industry in Japan. SC was degraded into low molecular weight fragments by a silk scouring process and released as a waste product.1) However, recent progress in transgenic technology has produced strains of transgenic silkworms that secrete recombinant proteins such as vaccines and cytokines in the SC of their cocoons. 7,8) Furthermore, the modification of silk protein properties by transgenic silkworms is now being attempted. Takabayashi reported the applicability of silk fibroin, expressed with a cell attachment factor, in the construction of biocompatible and biodegradable artificial blood vessels.9,10) Thus, modified silk protein as a functional material and a generator of recombinant protein is a promising new direction in the field of sericulture.In a study of the application of SC as a biomaterial, SC was reported to enhance the attachment of cultured human skin fibroblasts.11) In addition, SC hydrogel sheets containing a mixture of water ...

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

Cited by 74 publications

References 32 publications

Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori

Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori

Transgenic silkworms that weave recombinant proteins into silk cocoons

Use of Silk Protein, Sericin, as a Sustained-Release Material in the Form of a Gel, Sponge and Film

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