Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: Production of recombinant human serum albumin

Ogawa, Shingo; Tomita, Masahiro; Shimizu, Katsuhiko; Yoshizato, Katsutoshi

doi:10.1016/j.jbiotec.2006.10.019

Cited by 98 publications

(61 citation statements)

References 27 publications

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“…이 시스템은 벡터 및 헬퍼 플라스미드 DNA를 preblastodermal 누에알에 주입하여 형질전환누에를 제작 하는 방법으로서, 일본의 Tamura 연구팀이 나비목곤충인 Trichoplusia ni에서 유래한 piggyBac 전이벡터를 제작하 여 최초로 형질전환 누에 제작에 성공하였다 (Tamura et al 2000). 현재까지 piggyBac 전이벡터를 사용한 많은 누 에 형질전환 실험이 진행되었고, 이 발현 시스템을 사용 하여 basic fibroblast growth factor(bFGF), human serum albumin(HSA), feline interferon(FeIFN), insulin like growth factor-I(hIGF-I) 등이 성공적으로 발현된다고 보고되어 있 다 (Hino et al 2006, Kurihara et al 2007, Ogawa et al 2007, Tomita et al 2003, Zhao et al 2009). …”

Section: 서 론unclassified

Expression of the blue fluorescent protein in fibroin H-chain of transgenic silkworm

Kim¹,

Yun²,

Choi³

et al. 2014

Journal of Sericultural and Entomological Science

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We produced the transgenic silkworm that expressed the enhanced blue fluorescent protein (EBFP) in the cocoon of silkworms. The EBFP fusion protein, each with N-and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the EBFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3 × P3-driven DsRed2 cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. A mixture of the donor and helper vector was micro-injected into 300 eggs of silkworms, Baegokjam. We obtained 5 broods. The cocoon displayed blue fluorescence, proving that the fusion protein was present in the cocoon. Also, the presence of fusion proteins in cocoons was demonstrated by SDS-PAGE and western blot analysis. Accordingly, we suggest that the EBFP fluorescence silk will enable the production of the silk-based biomaterials.

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Section: 서 론unclassified

Expression of the blue fluorescent protein in fibroin H-chain of transgenic silkworm

Kim¹,

Yun²,

Choi³

et al. 2014

Journal of Sericultural and Entomological Science

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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.…”

mentioning

confidence: 99%

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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“…The cocoon is made mainly by silk fibroin, which is synthesized by the posterior silk gland (Hino et al, 2006;Ogawa et al, 2007). Silk fibroins consist of two subunits, fibroin H-and L-chains, which are linked by a disulfide-bond.…”

Section: Plasmid Dna Constructionmentioning

confidence: 99%

Expression of the cyan fluorescent protein in fibroin H-chain of transgenic silkworm

Goo¹,

Choi²,

Kim³

et al. 2017

International Journal of Industrial Entomology

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We constructed the fibroin H-chain expression system to produce enhanced cyan fluorescent proteins (ECFP) in transgenic silkworm cocoon. Fluorescent cocoon could be made by fusing ECFP cDNA to the heavy chain gene and injecting it into a silkworm. The ECFP fusion protein, each with N-and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the ECFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworms. The EGFP fluorescence became visible in the ocelli and in the central and peripheral nervous system on the seventh day of embryonic development. A mixture of the donor and helper vector was micro-injected into 1,020 Kumokjam, bivoltin silkworm eggs. We obtained 6 broods. The cocoon was displayed strong blue fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the ECFP fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.

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Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: Production of recombinant human serum albumin

Cited by 98 publications

References 27 publications

Expression of the blue fluorescent protein in fibroin H-chain of transgenic silkworm

Expression of the blue fluorescent protein in fibroin H-chain of transgenic silkworm

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

Expression of the cyan fluorescent protein in fibroin H-chain of transgenic silkworm

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