Expression of VEGFR2 Ligand Binding Domain in Pichia pink™ 4 Cells and Evaluation of Its Interactions with VEGF-A 165 Receptor Binding Domain.

Vascular endothelial growth factor A 165 (VEGF-A 165 ) and VEGF receptor 2 (KDR) are important mediators of angiogenesis. We aimed to express the soluble KDR ligand-binding domain (sKDR1-3) and evaluate its interaction with the VEGF-A 165 receptor-binding domain (VEGFA 165 -RBD). sKDR1-3 DNA was designed and subcloned into pPinkα-HC plasmid. The cassette was transfected into the Pichia pink™ 4 genome by homologous recombination. We optimized the expression of sKDR1-3 under the induction of different methanol concentrations. VEGFA 165 -RBD was expressed in E. coli BL21 harboring pET28a( +)─VEGFA 165 -RBD vector under induction with IPTG with/without lactose. Interaction and biological activity of sKDR1-3 and VEGFA 165 -RBD were investigated by ELISA and anti-proliferation tests. sKDR1-3 migrated on SDS-PAGE gel as a 35-180 kDa protein due to glycosylation. The relative expression level of sKDR1-3 under 1% methanol was higher than 0.5% and 4% methanol induction. IPTG and cysteine were suitable for induction and refolding of VEGFA 165 -RBD. 25 ng sKDR1-3 and 20 ng VEGFA 165 -RBD showed strong binding. sKDR1-3 bound to VEGFA 165 -RBD and VEGF-A 165 with dissociation constants of 0.148 and 0.2 nM, respectively. 4-10 nM concentrations of sKDR1-3 inhibited the proliferation of HUVE cells induced by 5 nM VEGFA 165 -RBD. In consideration, sKDR1-3 in the nanomolar concentration range, is a promising anticancer drug to inhibit angiogenesis.