title: Production of a non-triple helical collagen alpha chain in transgenic silkworms and its evaluation as a gelatin substitute for cell culture.
abstract: We generated transgenic silkworms that synthesized human type I collagen alpha1 chain [alpha1(I) chain] in the middle silk glands and secreted it into cocoons. The initial content of the recombinant alpha1(I) chain in the cocoons of the transgenic silkworms was 0.8%. The IE1 gene, a trans-activator from the baculovirus, was introduced into the transgenic silkworm to increase the content of the chain. We also generated silkworms homozygous for the transgenes. These manipulations increased the alpha1(I) chain content to 8.0% (4.24 mg per cocoon). The alpha1(I) chain was extracted and purified from the cocoons using a very simple method. The alpha1(I) chain contained no hydroxyprolines due to the absence of prolyl-hydroxylase activity in the silk glands. Circular dichroism analysis showed that the secondary structure of the alpha1(I) chain is similar to that of denatured type I collagen, demonstrating the absence of the triple helical structure. Human skin fibroblasts were seeded on the alpha1(I) chain-coated dishes. The cells attached and spread, although at decreased chain concentrations the spreading rate was lower than that of the collagen and gelatin. Cynomolgus monkey embryonic stem cells cultured on the alpha1(I) chain-coated dishes maintained an undifferentiated state after 30 passages, and their pluripotency was confirmed by teratoma formation in severe combined immunodeficient mice. These results show that the recombinant human alpha1(I) chain is a promising candidate biomaterial as a high-quality and safe gelatin substitute for cell culture.
authors.affiliation: Neosilk Co., Ltd., 3-13-26 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Adachi
authors.forename: Xiaobiao
authors.initials: X
authors.lastname: Wang
authors.forename: Tomoko
authors.initials: T
authors.lastname: Murata
authors.forename: Masanobu
authors.initials: M
authors.lastname: Obara
authors.forename: Hidenori
authors.initials: H
authors.lastname: Akutsu
authors.forename: Masakazu
authors.initials: M
authors.lastname: Machida
authors.forename: Akirihiro
authors.initials: A
authors.lastname: Umezawa
authors.forename: Masahiro
authors.initials: M
authors.lastname: Tomita
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D016367:Baculoviridae / Q000235:genetics; D012831:Bombyx / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002942:Circular Dichroism; D024042:Collagen Type I / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry*; D005347:Fibroblasts; D006801:Humans; D008252:Macaca fascicularis; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013234:Stem Cells; D015534:Trans-Activators / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
chemical_list: D024042:Collagen Type I; D003470:Culture Media; D011994:Recombinant Proteins; D015534:Trans-Activators; D014764:Viral Proteins