Characterization of the vascular endothelial growth factor-receptor interaction and determination of the recombinant protein by an optical receptor sensor
Characterization of the vascular endothelial growth factor-receptor interaction and determination of the recombinant protein by an optical receptor sensor
复制标题
DOI：
10.1016/s0956-5663(02)00090-8
复制
发表时间：
2002-12-01
影响因子：
12.6
通讯作者：
Bilitewski, U
中科院分区：
文献类型：
--
作者：
von Tiedemann, B;Bilitewski, U
血管内皮生长因子（VEGF）是控制血管生成的重要因子之一。它是属于半胱氨酸结蛋白家族的同型二聚体糖蛋白。生物活性通过酪氨酸激酶受体家族的跨膜受体转导。每个生物活性VEGF具有两个受体结合位点，导致受体二聚化作为配体结合后的第一步。受体的配体结合位点位于细胞外Ig样结构域上。将受体Flt-1（VEGFR-1）的细胞外部分表达为可溶性蛋白，并用作光学亲和传感器系统（BIAcore）中的受体。合适的条件允许测定结合和解离速率常数，分别为k（a）= 4 +/- 1.2 × 10（6）M-1 s（-1）和k（d）= 3 +/- 0.8 × 10（-5）s（-1），导致亲和常数K-D = 7.5 +/- 3 pM，这在其它研究和方法已经公布的范围内。增加传感器表面的受体负载降低了结合效率，因为结合的VEGF分子与理论上可用的结合位点的比率从1：1.5增加到1：2.6。进一步增加表面负载，允许建立定量测定，其中分析性能受到受体负载和样品与固定化受体之间的接触时间（即样品体积）的影响。该测定用于重组毕赤酵母菌株培养期间的VEGF测定。(C)2002 Elsevier Science B. V.保留所有权利。
Vascular endothelial growth factor (VEGF) is one of the most important factors controlling angiogenesis. It is a homodimeric glycoprotein belonging to the family of cysteine-knot proteins. The biological activity is transduced via membrane-spanning receptors of the tyrosine kinase receptor family. Each biologically active VEGF has two receptor binding sites leading to receptor dimerization as first step following ligand binding. The ligand-binding site of the receptor is localized on extracellular Ig-like domains. The extracellular part of the receptor Flt-1 (VEGFR-1) was expressed as soluble protein and was used as receptor in an optical affinity sensor system (BIAcore). Suitable conditions allowed the determination of the association and dissociation rate constants as k(a) = 4 +/- 1.2 x 10(6) M-1 s(-1) and k(d) = 3 +/- 0.8 x 10(-5) s(-1), respectively, leading to an affinity constant of K-D = 7.5 +/- 3 pM, which is within the range published already from other investigations and methods. Increasing receptor loadings of the sensor surface decreased the binding efficiency, as the ratio of bound VEGF-molecules to theoretically available binding sites increased from 1:1.5 to 1:2.6. Increasing the surface loading further, allowed the establishment of a quantitative assay with the analytical performance being influenced by the receptor loading and the contact time between sample and immobilized receptor, i.e. sample volume. This assay was used for VEGF determination during the cultivation of a recombinant Pichia pastoris strain. (C) 2002 Elsevier Science B.V. All rights reserved.