Production of a biologically active epidermal growth factor fusion protein with high collagen affinity.
Collagen is generally incapable of capturing polypeptides such as growth factors in a specific manner. In this study, we established a collagen-binding growth factor (FNCBD-EGF) consisting of epidermal growth factor (EGF) and the fibronectin collagen-binding domain. A typical yield of FNCBD-EGF was approximately 200 microg/ml culture in an Escherichia coli expression system. This fusion protein bound to gelatin and fibrillar collagen sponges, and the bound protein was not effectively eluted even with 2 M NaCl. In addition, FNCBD-EGF bound to type I, II, III, or IV collagen-coated plates, and the specificity of binding was confirmed by competitive inhibition using fibronectin. FNCBD-EGF substantially stimulated cell growth after binding to collagen-coated culture plates, whereas EGF had no effect, indicating that this fusion protein acted as a collagen-associated growth factor. In an animal model of impaired wound healing, FNCBD-EGF, but not EGF, was retained with collagen sponges at wound sites 4 d after implantation, and repair of epidermis was observed underneath the sponges. These results suggested that our fusion protein with high collagen affinity would be useful for wound healing.
Terumo Corporation R&D Center, Inokuchi, Nakai-machi, Ashigarakami-gun, Kanagawa 259-0151, Japan. tetsuya_ishikawa@terumo.co.jp
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Ishikawa
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Kitajima
D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003094:Collagen / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003920:Diabetes Mellitus / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D005780:Gelatin / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D013816:Thermodynamics; D014947:Wounds and Injuries / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D005780:Gelatin; D003094:Collagen