title: The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor.
abstract: 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.
authors.affiliation: Yoshizato Project, Cooperative Link of Unique Science and Technology for Economy Revitalization, Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan.
authors.forename: Rika
authors.initials: R
authors.lastname: Hino
authors.forename: Masahiro
authors.initials: M
authors.lastname: Tomita
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Yoshizato
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D012831:Bombyx / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D005349:Fibroins / Q000235:genetics / Q000378:metabolism*; D005854:Germ Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D047011:Silk / Q000737:chemistry*
chemical_list: D003470:Culture Media; D011993:Recombinant Fusion Proteins; D047011:Silk; D016222:Fibroblast Growth Factor 2; D005349:Fibroins