Expression of VEGFR2 Ligand Binding Domain in Pichia pink™ 4 Cells and Evaluation of Its Interactions with VEGF-A₁₆₅ Receptor Binding Domain

Vascular endothelial growth factor A₁₆₅ (VEGF-A₁₆₅) 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-A₁₆₅ receptor-binding domain (VEGFA₁₆₅-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. VEGFA₁₆₅-RBD was expressed in E. coli BL21 harboring pET28a( +)─VEGFA₁₆₅-RBD vector under induction with IPTG with/without lactose. Interaction and biological activity of sKDR1-3 and VEGFA₁₆₅-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 VEGFA₁₆₅-RBD. 25 ng sKDR1-3 and 20 ng VEGFA₁₆₅-RBD showed strong binding. sKDR1-3 bound to VEGFA₁₆₅-RBD and VEGF-A₁₆₅ 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 VEGFA₁₆₅-RBD. In consideration, sKDR1-3 in the nanomolar concentration range, is a promising anticancer drug to inhibit angiogenesis.