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The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor

Paper ID Volume ID Publish Year Pages File Format Full-Text
11221 725 2006 10 PDF Available
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 CaCl2, ethanol, and water at a molar ratio of 1:2:8 (CaCl2/ethanol/water) to solubilize fibroin layers. Western blot analysis showed that the recombinant protein, r(FL/bFGF), was solubilized with CaCl2/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.

Keywords
Silk; Transgenic silkworms; Human fibroblast growth factor; Bioactive fibroin; Scaffold; Human umbilical vein endothelial cells
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The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biomaterials - Volume 27, Issue 33, November 2006, Pages 5715–5724
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us