عنوان	Expression of VEGFR2 Ligand Binding Domain in Pichia pink™ 4 Cells and Evaluation of Its Interactions with VEGF-A165 Receptor Binding Domain
نوع پژوهش	مقاله چاپ‌شده در مجلات علمی
کلیدواژه‌ها	VEGF-A165; VEGFR2; Pichia pinkTM 4 cells; ELISA; Heparin binding domain; Affinity constant; Angiogenesis; HUVE cells
چکیده	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 pinkTM 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.
پژوهشگران	زهرا فتحی (نفر اول)، مسعود مشهدی اکبر بوجار (نفر دوم)، رضا حسن ساجدی (نفر سوم)، احسان دهنوی (نفر چهارم)، زهره جهان افروز (نفر پنجم)