Production of a non‐triple helical collagen α 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.

Mussel-inspired human gelatin nanocoating for creating biologically adhesive surfaces

“…The rhG was produced by transgenic silkworms and purified from their cocoons in the manner described by Adachi et al 7 A fusion protein consisting of the fibronectin collagen binding domain and vascular endothelial growth factor (CBD-VEGF) was prepared as described previously. 39 …”

“…3 Olsen et al prepared low molecular weight recombinant human gelatin using yeast, 4 Duan et al reported an expression system for hydroxylated gelatin using Pichia pastoris, 5 Shoseyov et al developed a technique to generate recombinant human collagen from a genetically engineered tobacco plant, 6 and Adachi et al used transgenic silkworms to produce recombinant human gelatin by introducing a type I collagen α1 chain gene that encompassed the entire triple helical region. 7 We have modified recombinant human gelatin to expand its functions and applications. For example, gelatin modified with cholesterol was prepared as nanosized gel particles, 8,9 and photoreactive gelatin was prepared to covalently immobilize gelatin itself and to coimmobilize other biological molecules on various types of organic materials.…”

Differential response of arterial and venous endothelial cells to extracellular matrix is modulated by oxygen

“…Consequently, the extracellular matrix (ECM) is one of the most important factors determining the endothelial cell properties in vivo and in vitro (Adachi et al 2010;Kuehn and Timpl 1982;Madri and Pratt 1986). Oxygen tension varies within the vasculature (Adachi et al 2010;Read et al 1995) and thus can also affect a broad range of biological functions, i.e. cell proliferation, apoptosis, inflammation and angiogenesis (Simon and Keith 2008;Weidemann and Johnson 2008).…”

“…The endothelial cell has a luminal surface making contact with the blood, and an abluminal surface in close interaction with the subendothelial basement membrane. Consequently, the extracellular matrix (ECM) is one of the most important factors determining the endothelial cell properties in vivo and in vitro (Adachi et al 2010;Kuehn and Timpl 1982;Madri and Pratt 1986). Oxygen tension varies within the vasculature (Adachi et al 2010;Read et al 1995) and thus can also affect a broad range of biological functions, i.e.…”

Positively charged cholesterol–recombinant human gelatins foster the cellular uptake of proteins and murine immune reactions

“…This overcomes the above limitations as it is homogeneous in MW, nontoxic, and is being considered as an alternative to conventional gelatin extracted from bovine or porcine sources. 17,18 We report the synthesis of two types of rhG derivatives by using two kinds of cholesterol derivatives. They have the ability to form micelles but differ in terms of their zeta (ζ)-potential.…”