title: Surface immobilization of active vascular endothelial growth factor via a cysteine-containing tag.
abstract: Developing tissue engineering scaffolds with immobilized growth factors requires facile and reliable methods for the covalent attachment of functionally active proteins. We describe here a new approach to immobilize recombinant proteins based on expression of the protein of interest with a 15-aa long fusion tag (Cys-tag), which avails a free sulfhydryl group for site-specific conjugation. To validate this approach, we conjugated a single-chain vascular endothelial growth factor expressed with an N-terminal Cys-tag (scVEGF) to fibronectin (FN) using a common thiol-directed bi-functional cross-linking agent. We found that the FN-scVEGF conjugate retains VEGF activity similar to that of free scVEGF when used as a soluble ligand. Cells expressing VEGF receptor VEGFR-2 grown on plates coated with FN-scVEGF displayed morphological phenotypes similar to those observed for cells grown on FN in the presence of equivalent amounts of free scVEGF. In addition, 293/KDR cell growth stimulation was observed in the same concentration range with either immobilized or free scVEGF. The effects of immobilized scVEGF, and soluble scVEGF were blocked by NVP-AAD777-NX, a VEGF receptor tyrosine kinase inhibitor. These data indicate that site-specific immobilization via Cys-tag provides a facile and reliable method for permanent deposition of functionally active growth factors on synthetic or protein scaffolds with applications for advanced tissue engineering.
authors.affiliation: SibTech, Inc., Newington, CT 06111, USA. mbacker@sibtech.com
authors.forename: Marina V
authors.initials: MV
authors.lastname: Backer
authors.forename: Vimal
authors.initials: V
authors.lastname: Patel
authors.forename: Brian T
authors.initials: BT
authors.lastname: Jehning
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Claffey
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Backer
mesh_terms: D001665:Binding Sites; D002448:Cell Adhesion; D002460:Cell Line; D049109:Cell Proliferation; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D003460:Crystallization / Q000379:methods*; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D007668:Kidney / Q000166:cytology* / Q000378:metabolism*; D011485:Protein Binding; D023822:Tissue Engineering / Q000379:methods*; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism
chemical_list: D020099:Coated Materials, Biocompatible; D003432:Cross-Linking Reagents; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D003545:Cysteine