A fusion protein of hepatocyte growth factor for immobilization to collagen.
We describe here a fusion protein consisting of hepatocyte growth factor (HGF; an angiogenic factor) and a collagen-binding domain (CBD) polypeptide of fibronectin (FN). This fusion protein (CBD-HGF), produced by a baculovirus expression system, exhibited much stronger collagen binding activity than native HGF in the range of 0.4-6.4microg/ml. Its binding at the lowest concentration exceeded that of HGF at the highest concentration. In addition, the collagen-bound CBD-HGF promoted growth of endothelial cells (ECs) to a greater degree at least 4 days longer than HGF added to the culture medium; about 5-fold greater increase in cell number after 10 days. These findings suggest that the fused CBD moiety not only helped immobilize HGF on collagen but also helped stabilize the fusion molecule, resulting in prolonged activity. The angiogenic activity of CBD-HGF in animal tissues was examined by subcutaneously implanting collagen sponges containing bound CBD-HGF. Blood vessel formation in the sponges after 7 days was 4-6-fold extensive as compared to the control sponges without sample. Implanted sponges with native HGF did not show significant difference from control. These results indicate that CBD-HGF is suitable for in vitro culture of ECs, and that this fusion protein can be used to confer HGF activity on biomaterials for use in tissue engineering.
Kitajima
Takashi
T
Regenerative Medical Bioreactor Project, Kanagawa Academy of Science and Technology, KSP East 309, Sakado 3-2-1, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan. vyb04604@nifty.com
Terai
Hiroshi
H
Ito
Yoshihiro
Y
D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry*; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D009389:Neovascularization, Pathologic; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012038:Regeneration; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods
D001672:Biocompatible Materials; D005353:Fibronectins; D011993:Recombinant Fusion Proteins; D017228:Hepatocyte Growth Factor; D003094:Collagen