Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134, Naples, Italy.
Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Università della Campania "L. Vanvitelli", Via Vivaldi, 43 - 81100, Caserta, Italy.
Mol Biotechnol. 2019 Nov;61(11):860-872. doi: 10.1007/s12033-019-00211-4.
Angiogenesis is a biological process finely tuned by a plethora of pro- and anti-angiogenic molecules, among which vascular endothelial growth factors (VEGFs). Their biological activity is expressed through the interaction with three cognate receptor tyrosine kinases, VEGFR1, 2, and 3. VEGFR2 is the primary regulator of angiogenesis. Ligand-induced VEGFR2 dimerization and activation depend on direct ligand binding to extracellular domains 2 and 3 of receptor and in the establishment of interactions between proximal membrane domains. VEGFR2 domain 7 has been shown to play a crucial role in receptor dimerization and regulation, therefore, representing a convenient target for the allosteric modulation of VEGFR2 activity. The ability to prepare a functional VEGFR2D7 domain represents the starting point to the development of novel VEGFR2 binders acting as allosteric inhibitors of receptor activity. Here, we describe a robust and efficient procedure for the preparation in E. coli of the VEGFR2 domain 7. The protein was obtained with a good yield and was properly folded. It was investigated in a biochemical and structural study, providing information on its conformational arrangement and in solution properties.
血管生成是一个由大量促血管生成和抗血管生成分子精细调控的生物学过程，其中包括血管内皮生长因子（VEGFs）。它们的生物活性通过与三个同源受体酪氨酸激酶（VEGFR1、2 和 3）的相互作用来表达。VEGFR2 是血管生成的主要调节因子。配体诱导的 VEGFR2 二聚化和激活依赖于受体的细胞外结构域 2 和 3 与近膜结构域之间的直接配体结合和相互作用。已经表明 VEGFR2 结构域 7 在受体二聚化和调节中起着至关重要的作用，因此，它是 VEGFR2 活性变构调节的一个方便的靶点。制备功能性 VEGFR2D7 结构域的能力代表了开发新型 VEGFR2 结合物作为受体活性变构抑制剂的起点。在这里，我们描述了一种在大肠杆菌中制备 VEGFR2 结构域 7 的稳健且高效的程序。该蛋白以良好的产率获得并正确折叠。它在生化和结构研究中进行了研究，提供了有关其构象排列和溶液性质的信息。
