title: Engineering, expression, and renaturation of a collagen-targeted human bFGF fusion protein.
abstract: Basic fibroblast growth factor (bFGF) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and may participate in tissue repair. Basic FGF is found in abundance in tissues such as brain, kidney and cartilage. This study reports the expression, purification, and renaturation of a biologically active human basic fibroblast growth factor fusion protein (hbFGF-F1) from Escherichia coli. A prokaryotic expression vector was engineered to produce a tripartite fusion protein consisting of (i) a purification tag, (ii) a protease-sensitive linker/collagen-binding domain, and (iii) cDNA sequence encoding the active fragment of hbFGF. The expressed hbFGF-F1 and hbFGF-F2 (it contains a collagen-binding domain), located in inclusion bodies, were solubilized with 6 M guanidine-HCl and renatured using a glutathione redox system and protracted dialysis under various experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion protein on a Ni-NTA metal chelate column. The biological activity of the recombinant growth factors was demonstrated by their ability to stimulate proliferation of human vein endothelial cells (HVEC), monitored by [3H]-thymidine incorporation, where commercial recombinant human bFGF (rhbFGF) served as a positive control. Purified rhbFGF-F1 and rhbFGF-F2 constructs exhibited proliferative activity comparable to commercial rhbFGF. Binding of the renatured hbFGF-F2 fusion protein to collagen was demonstrated by stable binding on a collagen-conjugated Sephadex-G15 column. The high affinity binding was also demonstrated by the binding of [3H]-collagen to the rhbFGF-F2 protein immobilized on a Ni-NTA column. The rhbFGF-F2 fusion protein bound to collagen coated surfaces with high affinity but exhibited comparatively lower biological activity than the fusion protein in solution, suggesting a potentially latent configuration. Taken together, these results demonstrate that biologically active rhbFGF fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for its high-yield production, purification, and renaturation from microorganisms. Furthermore, we demonstrate that the auxiliary collagen-binding domain effectively targets the recombinant growth factor to type I collagen. The clinical effect of rhbFGF-F2 on wound healing is also studied in streptozotocin-induced diabetic rats and evaluated by histological examination comparing with rhbFGF-F1 and commercial bFGF effects. The highly beneficial effects of rhbFGF-F2 on wound healing is suggested to be due to its extremely potent angiogenesis and granulation tissue formation activities, leading to a rapid reepithelialization of the wound. Topical application of rhbFGF-F2 mixed with type I collagen is a more effective method in accelerating closure of full-thickness excisional skin-wound in diabetic rats when compared with the fusion protein alone or commercial hbFGF at the same doses. These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins as well as to develop new strategies for specific biomedical applications.
authors.affiliation: Department of Cell Biology and Genetics, Faculty of Sciences, University of Málaga, Spain.
authors.forename: J A
authors.initials: JA
authors.lastname: Andrades
authors.forename: L T
authors.initials: LT
authors.lastname: Wu
authors.forename: F L
authors.initials: FL
authors.lastname: Hall
authors.forename: M E
authors.initials: ME
authors.lastname: Nimni
authors.forename: J
authors.initials: J
authors.lastname: Becerra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D008297:Male; D015202:Protein Engineering; D020673:Protein Renaturation; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012995:Solubility; D014945:Wound Healing / Q000187:drug effects
chemical_list: D011993:Recombinant Fusion Proteins; D016222:Fibroblast Growth Factor 2; D003094:Collagen