Purification and refolding of vascular endothelial growth factor-B.
Vascular endothelial growth factor (VEGF)-A interacts with the receptor tyrosine kinases VEGF-R1 and R2, and the importance of this interaction in endothelial cell (EC) function and blood vessel development has been well documented. Other ligands that interact differentially with these receptors and that are structurally related to VEGF-A include VEGF-B, VEGF-C, VEGF-D, and placenta growth factor (PLGF). Compared with VEGF-A, relatively little is known about the biological role of the VEGF-R1 specific ligand, VEGF-B. Two splice variant isoforms that differ at the COOH-terminus and which retain unique solubility characteristics are widely expressed throughout embryonic and postnatal development. Recent analysis of mice with a targeted deletion of the VEGF-B gene has revealed a defect in heart development and function consistent with an important role in vascularization of the myocardium (Bellomo D et al., 2000, Circ Res 86:E29-E35). To facilitate further characterization of VEGF-B, we have developed a protocol for expression and purification of refolded recombinant protein from Escherichia coli inclusion bodies (IBs). The approach developed resolves a number of significant issues associated with VEGF-B, including the ability to heterodimerize with endogenous VEGF-A when co-expressed in mammalian cells, a complex secondary structure incorporating inter- and intrachain disulfide bonds and hydrophobic characteristics that preclude the use of standard chromatographic resins. The resulting purified disulfide-linked homodimer was demonstrated to bind to VEGF-R1 and to compete with VEGF-A for binding to this receptor.
Scrofani
S D
SD
AMRAD Operations Pty Ltd, Richmond, Victoria, Australia.
Fabri
L J
LJ
Xu
P
P
Maccarone
P
P
Nash
A D
AD
D000818:Animals; D015374:Biosensing Techniques; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D019281:Dimerization; D016228:Endothelial Growth Factors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D011489:Protein Denaturation; D017510:Protein Folding; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000737:chemistry / Q000378:metabolism; D040262:Receptors, Vascular Endothelial Growth Factor; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance; D042461:Vascular Endothelial Growth Factor A; D042561:Vascular Endothelial Growth Factor B; D040281:Vascular Endothelial Growth Factor Receptor-1
D016228:Endothelial Growth Factors; D011518:Proto-Oncogene Proteins; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042561:Vascular Endothelial Growth Factor B; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D040281:Vascular Endothelial Growth Factor Receptor-1