Purification and refolding of vascular endothelial growth factor-B

Vascular endothelial growth factor (VEGF)-A interacts with the receptor tyr osine kinases VEGF-R1 and R2, and the importance of this interaction in end othelial cell (EC) function and blood vessel development has been well docu mented. Other ligands that interact differentially with these receptors and that are structurally related to VEGF-A include VEGF-B, VEGF-C, VEGF-D, an d placenta growth factor (PLGF). Compared with VEGF-A, relatively little is known about the biological role of the VEGF-R1 specific ligand, VEGF-B. Tw o splice variant isoforms that differ at the COOH-terminus and which retain unique solubility characteristics are widely expressed throughout embryoni c and postnatal development. Recent analysis of mice with a targeted deleti on of the VEGF-B gene has revealed a defect in heart development and functi on consistent with an important role in vascularization of the myocardium ( Bellomo D et al., 2000, Circ Res 86:E29-E35). To facilitate further charact erization of VEGF-B, we have developed a protocol for expression and purifi cation of refolded recombinant protein from Escherichia coli inclusion bodi es (IBs). The approach developed resolves a number of significant issues as sociated with VEGF-B, including the ability to heterodimerize with endogeno us VEGF-A when co-expressed in mammalian cells, a complex secondary structu re incorporating inter- and intrachain disulfide bonds and hydrophobic char acteristics that preclude the use of standard chromatographic resins. The r esulting purified disulfide-linked homodimer was demonstrated to bind to VE GF-R1 and to compete with VEGF-A for binding to this receptor.