Expression Of KDR Tyrosine Kinase And Screening Of Its Inhibitors

Flk-1/KDR is the most important specific receptor of vascular endothelial growth factor, which can mediate biological functions and effects of vascular endothelial cell proliferation. The formation of new vascular is the essential factor of malignant tumor growth, so using KDR tyrosine kinase as a target for drug design and screening has become an important strategy of anti-tutor.The main objective of this study is to establish a high-throughput screening model for inhibitors of KDR tyrosine kinase with purified KDR tyrosine kinase from recombined E. coli. The KDR tyrosine kinase was expressed in E. coli M15 by plasmid pQE30 as vector. The soluble expression level of KDR tyrosine kinase could be promoted when cells were cultured at 22℃for 3h to induce with IPTG of 0.5mmol/L. The recombinant protein was purified with affinity chromatography (Ni-NTA), the purity and concentration of recombinant protein had reached experimental requirements. SDS-PAGE analysis showed that the molecular mass of expressed KDR tyrosine kinase was about 36kDa as expected. Western blot analysis indicated that the recombinant protein could react specifically against both anti-Flk-1 and anti-His antibody. Future analysis showed that the recombinant protein had autophosphorylation activity. Using the KDR tyrosine kinase as target, the screening models for enzymatic inhibitors were constructed by ELISA and AlphaScreen. SU11248 and PTK787, the known KDR tyrosine kinase inhibitor, were used as the positive control. The results demonstrated that two models both effectively identified known KDR tyrosine kinase inhibitors. The assay conditions of two methods were optimized and they were compared with respect to kinetics and correlation. The data showed that the two models had significant correlation, but AlphaScreen was short time consuming. Finally, a cobalt-contained compound which had anti-tumor activity was selected to test the model and found that the compounds could inhibit KDR tyrosine kinase effectively.