title: Coiled-coil-mediated grafting of bioactive vascular endothelial growth factor.
abstract: Chimeric growth factors may represent a powerful alternative to their natural counterparts for the functionalization of tissue-engineered scaffolds and applications in regenerative medicine. Their rational design should provide a simple, readily scalable production strategy while improving retention at the site of action. In that endeavor, we here report the synthesis of a chimeric protein corresponding to human vascular endothelial growth factor 165 being N-terminally fused to an E5 peptide tag (E5-VEGF). E5-VEGF was successfully expressed as a homodimer in mammalian cells. Following affinity purification, in vitro surface plasmon resonance biosensing and cell survival assays confirmed diffusible E5-VEGF ability to bind to its receptor ectodomains, while observed morphological phenotypes confirmed its anti-apoptotic features. Additional surface plasmon resonance assays highlighted that E5-VEGF could be specifically captured with high stability when interacting with covalently immobilized K5 peptide (a synthetic peptide designed to bind to the E5 moiety of chimeric hVEGF). This immobilization strategy was applied to glass substrates and chimeric hVEGF was shown to be maintained in a functionally active state following capture. Altogether, our data demonstrated that stable hVEGF capture can be performed via coiled-coil interactions without impacting hVEGF bioactivity, thus opening up the way to future applications in the field of tissue engineering and regenerative medicine.
authors.affiliation: Department of Chemical Engineering, Biomedical Science and Technology Research Group, Bio-P(2) Research Unit, École Polytechnique de Montréal, P.O. Box 6079, succ. Centre-Ville, Montréal (QC), Canada H3C 3A7.
authors.forename: Frederic
authors.initials: F
authors.lastname: Murschel
authors.forename: Benoit
authors.initials: B
authors.lastname: Liberelle
authors.forename: Gilles
authors.initials: G
authors.lastname: St-Laurent
authors.forename: Mario
authors.initials: M
authors.lastname: Jolicoeur
authors.forename: Yves
authors.initials: Y
authors.lastname: Durocher
authors.forename: Gregory
authors.initials: G
authors.lastname: De Crescenzo
mesh_terms: D001665:Binding Sites; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017978:Receptors, Growth Factor / Q000737:chemistry* / Q000648:ultrastructure*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000235:genetics / Q000648:ultrastructure; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*
chemical_list: D017978:Receptors, Growth Factor; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D049452:Green Fluorescent Proteins