Huang X, Gottstein C, Brekken R A, Thorpe P E
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, 75235, USA.
Biochem Biophys Res Commun. 1998 Nov 27;252(3):643-8. doi: 10.1006/bbrc.1998.9717.
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.
血管内皮生长因子（VEGF）是一种内皮细胞特异性促有丝分裂原，在多种病理条件下可诱导血管生成。为了阻断血管生成，可使用可溶性VEGF受体。在本研究中，我们描述了一种在杆状病毒表达系统中高产表达可溶性VEGF受体2（sFlk-1）的方法（每升昆虫细胞上清液可纯化得到30 mg sFlk-1）。我们还通过表面等离子体共振测定了重组受体与人及小鼠VEGF的结合常数。在这种无细胞测定中，在给定的实验条件下，结合速率常数ka为0.5 - 2.2×10⁶ M⁻¹s⁻¹，解离速率常数kd为2 - 4×10⁻⁴ s⁻¹（KD = 2 - 6×10⁻¹⁰ M）。据我们所知，这是第一项报道VEGF与sFlk-1相互作用的结合和解离速率的研究。在该测定中，VEGF与sFlk-1的结合不需要肝素。所获得的值将作为设计重组可溶性VEGF受体改进版本的基线参数。
