Zhang Juan, Zhang Yanli, Li Haixin, Chen Wei, Zhang Tao, Wang Min
Department of Molecular Biology, School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, China.
Sheng Wu Gong Cheng Xue Bao. 2008 Nov;24(11):1962-7.
Vascular Endothelial Growth Factor Receptor-2(VEGFR-2) plays an important role in stimulating the proliferation of endothelial cells and improving the permeability of blood vessel. We prepared recombinant extracellular domain 3 (KDR3) of human vascular endothelial growth factor receptor-2 in Escherichia coli and studied its specific binding activity with its ligand. The target DNA was synthesized by overlapping PCR, ligated with expression vector p-ET32a and transformed into E. coli Rosetta (DE3). The soluble fusion protein Trx-KDR3 was expressed in cytoplasm, which was up to 20% of total soluble protein in cytoplasm after having been induced by 1 mmol/L IPTG for 5 h at 30 degrees C. It was characterized to be target protein by Western blotting. The product was purified by CM cation exchange resin and immobilized metal affinity chromatography(IMAC). Its VEGF-binding activity was determined by ELISA assay and its influence on the propagation of HUVEC induced by VEGF. The protein product showed high ligand binding activity in the ELISA and HUVEC propagation study compared to the control. Therefore, ligand binding active, soluble recombinant extracellular domain 3 of VEGFR-2 KDR was successfully expressed and purified in E. coli, which would be applied to anti-angiogenesis anti-tumor therapy and anti-KDR antibody development.
血管内皮生长因子受体-2（VEGFR-2）在刺激内皮细胞增殖和提高血管通透性方面发挥着重要作用。我们在大肠杆菌中制备了人血管内皮生长因子受体-2的重组胞外结构域3（KDR3），并研究了其与配体的特异性结合活性。通过重叠PCR合成靶DNA，将其与表达载体p-ET32a连接并转化到大肠杆菌Rosetta（DE3）中。可溶性融合蛋白Trx-KDR3在细胞质中表达，在30℃下用1 mmol/L IPTG诱导5小时后，其表达量占细胞质中总可溶性蛋白的20%。通过蛋白质印迹法鉴定其为靶蛋白。产物通过CM阳离子交换树脂和固定化金属亲和色谱（IMAC）进行纯化。通过ELISA测定其VEGF结合活性，并研究其对VEGF诱导的人脐静脉内皮细胞（HUVEC）增殖的影响。与对照相比，该蛋白产物在ELISA和HUVEC增殖研究中显示出高配体结合活性。因此，VEGFR-2 KDR的具有配体结合活性的可溶性重组胞外结构域3在大肠杆菌中成功表达并纯化，可应用于抗血管生成抗肿瘤治疗和抗KDR抗体的开发。
