Expression of Soluble VEGF Receptor 2 and
Characterization of Its Binding by Surface
Xianming Huang,1Claudia Gottstein, Rolf A. Brekken, and Philip E. Thorpe
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235
Received October 21, 1998
Vascular endothelial growth factor (VE GF ) is an en-
dothelial cell specific mitogen that induces angiogen-
esis in several pathological conditions. T o block angio-
genesis, soluble VE GF receptor can be used. In this
study, we describe a method for high yield expression
of soluble VE GF receptor 2 (sF lk-1) in a baculovirus
expression system (30 mg purified sF lk-1 per L of in-
sect cell supernatant). We also determined the binding
constants for both human and mouse VE GF to the
recombinant receptor by surface plasmon resonance.
In this cell-free assay, under the given experimental
conditions, the on-rate kawas 0.5-2.2 x 106M?1s?1and
the off-rate kdwas 2-4 x 10?4s?1(KD? 2-6 x 10?10M).
T o our knowledge this is the first study to report on-
and off-rates for the VE GF :sF lk-1 interaction. Heparin
was not required for the binding of VE GF to sF lk-1 in
this assay. T he obtained values will serve as baseline
parameters for the design of improved versions of re-
combinant soluble VE GF receptor.
© 1998 Academic Press
Vascular endothelial growth factor (VEGF) and its
receptors VEGFR1 (Flt-1, fms-like tyrosine kinase)
and VEGFR2 (KDR/Flk-1, kinase insert domain-
containing receptor/fetal liver kinase) have recently
attracted considerable interest because of their in-
volvement in vasculogenesis and angiogenesis (1–9).
Both VEGF and its receptors are overexpressed in a
number of angiogenesis dependent diseases such as
malignant tumors and diabetic retinopathy. The inhi-
bition of VEGF-mediated signals could thereforeoffer a
valuable approach for the therapy of these diseases.
Inhibition can be achieved through neutralizing anti-
bodies and soluble receptor constructs which block the
activation of VEGF receptors by preventing either li-
gand binding or ligand induced dimerization of recep-
tors. Previous reports have demonstrated that both
neutralizing VEGF and anti-KDR antibodies as well
as soluble VEGF receptor constructs are capable of
reducing or inhibiting tumor growth in animal models
It is now necessary to develop methods for express-
ing recombinant soluble VEGF receptor in high yields
for extensive preclinical and clinical testing. In this
report, we describe the expression of sFlk-1 in a bacu-
lovirus system and its purification by affinity chroma-
tography. The yield of highly purified receptor after a
single purification step was 30 mg/L supernatant.
Surface plasmon resonance was used to determine
the binding of the soluble recombinant receptor in real
time to its ligand VEGF. On- and off-rates for the
VEGF:sFlk-1 interaction, and the effect of heparin on
this interaction are reported.
MATERIALS AND METHODS
1. Construction of plasmids.
provided by Dr. Ihor Lemischka (Princeton University). This con-
struct contains the full length mouse Flk-1 cDNA. Double stranded
DNA encoding for the first 762 amino acids of Flk-1 was obtained by
PCR reaction using the primers GCACAGCTGATGGAGAGCAAG-
GCGCTGCTA and CTGCAGCTGCTATTCCAAGTTGGTCTTTTC
with the Flk-1 cDNA construct as a template. The resulting 2.3 kB
PCR fragment was cloned into pVL1393 (Pharmingen) and into
pBlueBacHis (InVitrogen). The DNA sequence was verified by auto-
mated fluorescence sequencing.
A Flk-1 cDNA construct was kindly
2. Expression of soluble Flk-1.
cultures of Spodoptera frugiperda (Sf9) cells were grown in Sf900II-
SFM (Gibco/BRL) supplemented with 50 ?g/ml penicillin and 50
?g/ml streptomycin (Gibco/BRL). Recombinant plasmids pVL1393/
sFlk-1 and pBlueBacHis/sFlk-1 were cotransformed with baculovi-
rus gold DNA (Pharmingen) intoSf9 cells by using lipofectin (Gibco/
BRL) according tothe manufacturer’s instructions. The recombinant
virus was plaque purified as described (18). To confirm the expres-
sion of recombinant protein, aliquots of conditioned medium of in-
fected Sf9 cells were analyzed by Western Blot using the monoclonal
antibody 1A8 directed against sFlk-1 (Brekken et al.; manuscript in
Monolayer and suspension cell
1To whom correspondence should be addressed at present ad-
dress: Maine Medical Center Research Institute, South Portland,
ME. Fax: (207) 761-9782. E-mail: HuangX@mail.mmc.org.
Abbreviations used: sFlk-1, soluble fetal liver kinase 1; VEGF121,
VEGF 165, vascular endothelial growth factor, 121 or 165 amino
acids length (splice variants); VEGFR, vascular endothelial growth
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 252, 643–648 (1998)
ARTICLE NO. RC989717
Copyright © 1998 by Academic Press
All rights of reproduction in any form reserved.
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