Expression of soluble VEGF receptor 2 and characterization of its binding by surface plasmon resonance.

Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that induces angiogenesis in several pathological conditions. To block angiogenesis, soluble VEGF receptor can be used. In this study, we describe a method for high yield expression of soluble VEGF receptor 2 (sFlk-1) in a baculovirus expression system (30 mg purified sFlk-1 per L of insect cell supernatant). We also determined the binding constants for both human and mouse VEGF to the recombinant receptor by surface plasmon resonance. In this cell-free assay, under the given experimental conditions, the on-rate ka was 0.5-2.2 x 10(6) M-1s-1 and the off-rate kd was 2-4 x 10(-4) s-1 (KD = 2-6 x 10(-10) M). To our knowledge this is the first study to report on- and off-rates for the VEGF:sFlk-1 interaction. Heparin was not required for the binding of VEGF to sFlk-1 in this assay. The obtained values will serve as baseline parameters for the design of improved versions of recombinant soluble VEGF receptor.

[1]  M. Dewhirst,et al.  Inhibition of tumor growth by targeting tumor endothelium using a soluble vascular endothelial growth factor receptor. , 1998, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[2]  Bing Li,et al.  Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo , 1993, Nature.

[3]  A. Minton,et al.  Analysis of mass transport-limited binding kinetics in evanescent wave biosensors. , 1996, Analytical biochemistry.

[4]  M. Longaker,et al.  Regulation of Vascular Endothelial Growth Factor Expression in Cultured Keratinocytes. , 1995, The Journal of Biological Chemistry.

[5]  Hung V. Nguyen,et al.  The Carboxyl-terminal Domain(111165) of Vascular Endothelial Growth Factor Is Critical for Its Mitogenic Potency (*) , 1996, The Journal of Biological Chemistry.

[6]  H. Dvorak,et al.  Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis , 1994, The Journal of experimental medicine.

[7]  Janet Rossant,et al.  Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice , 1995, Nature.

[8]  Leonard,et al.  Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. , 1997, Cancer research.

[9]  S. Kondo,et al.  Inhibition of tumor growth and metastasis by an immunoneutralizing monoclonal antibody to human vascular endothelial growth factor/vascular permeability factor121. , 1995, Cancer research.

[10]  R. Linhardt,et al.  Identification of a heparin binding peptide on the extracellular domain of the KDR VEGF receptor. , 1997, Growth Factors.

[11]  J. Stone,et al.  Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity , 1995, Nature Medicine.

[12]  S. Kondo,et al.  Significance of vascular endothelial growth factor/vascular permeability factor for solid tumor growth, and its inhibition by the antibody. , 1993, Biochemical and biophysical research communications.

[13]  K. Hillan,et al.  Complete inhibition of angiogenesis and growth of microtumors by anti-vascular endothelial growth factor neutralizing antibody: novel concepts of angiostatic therapy from intravital videomicroscopy. , 1996, Cancer research.

[14]  K. Plate,et al.  Vascular endothelial growth factor and glioma angiogenesis: Coordinate induction of VEGF receptors, distribution of VEGF protein and possible In vivo regulatory mechanisms , 1994, International journal of cancer.

[15]  A Mathis,et al.  Detection of vascular endothelial growth factor messenger RNA and vascular endothelial growth factor-like activity in proliferative diabetic retinopathy. , 1994, Archives of ophthalmology.

[16]  J. Flanagan,et al.  Interactions between the Flk-1 receptor, vascular endothelial growth factor, and cell surface proteoglycan identified with a soluble receptor reagent. , 1995, Growth factors.

[17]  D. Hicklin,et al.  Heparin modulates the interaction of VEGF165 with soluble and cell associated flk-1 receptors. , 1994, The Journal of biological chemistry.

[18]  E. Manseau,et al.  Expression of vascular permeability factor/vascular endothelial growth factor by melanoma cells increases tumor growth, angiogenesis, and experimental metastasis. , 1996, Cancer research.

[19]  D. Lane,et al.  An immunoaffinity purification procedure for SV40 large T antigen. , 1985, Virology.

[20]  R. Kendall,et al.  Specificity of vascular endothelial cell growth factor receptor ligand binding domains. , 1994, Biochemical and biophysical research communications.

[21]  K. Lehmann-Bruinsma,et al.  Vascular endothelial growth factor causes endothelial proliferation after vascular injury. , 1995, Biochemical and biophysical research communications.

[22]  J. Kaplan,et al.  Characterization of a soluble vascular endothelial growth factor receptor-immunoglobulin chimera. , 1997, Growth factors.

[23]  H. Dvorak,et al.  Inhibition of vascular permeability factor (vascular endothelial growth factor) with antipeptide antibodies. , 1993, Archives of biochemistry and biophysics.

[24]  S. Soker,et al.  The binding of vascular endothelial growth factor to its receptors is dependent on cell surface-associated heparin-like molecules. , 1992, The Journal of biological chemistry.

[25]  D. Gospodarowicz,et al.  VEGF receptor subtypes KDR and FLT1 show different sensitivities to heparin and placenta growth factor. , 1994, Growth factors.

[26]  J. Rossant,et al.  Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium , 1995, Nature.

[27]  Mihaela Skobe,et al.  Halting angiogenesis suppresses carcinoma cell invasion , 1997, Nature Medicine.