Volume 11, Issue 1 (2020)                   JMBS 2020, 11(1): 93-102 | Back to browse issues page

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Fathi Z, Mashhadi Akbar Boojar M, Dehnavi E, Hassan Sajedi R. Cloning and Expression of Extracellular Domain of Human VEGF Receptor-2 in Pichia pastoris and Investigation of it's Binding Properties. JMBS 2020; 11 (1) :93-102
URL: http://biot.modares.ac.ir/article-22-32940-en.html
1- Department of Cellular and Molecular Sciences, Faculty of Biological Sciences, Kharazmi University
2- Gene Transfer Pioneers Research Group, Shahid Beheshti University of Medical Science
3- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University , sajedi_r@modares.ac.ir
Abstract:   (3449 Views)
Abnormal angiogenesis is associated with various diseases such as solid tumors and metastasis, retinopathies, and rheumatoid arthritis. VEGF-A is the most important mediator of angiogenesis among all growth factors. The bioactivity of VEGF is mediated by two tyrosine kinase receptors VEGFR-1 and VEGFR-2 present on endothelial cells. VEGF signaling through VEGFR-2 is the major angiogenic pathway that leads to stimulation of endothelial cell ingrowths into the tumor. It comprised of an extracellular portion, a cytoplasmic portion, and a short transmembrane domain. The extracellular portion consists of seven Ig-like domains (D1–D7), of which the 1st to 3rd domains function as ligand-binding sites. In the present work, a soluble recombinant extracellular domain 1-3 of VEGFR-2 was expressed in Pichia pastoris to inhibit the VEGF-induced angiogenesis. The 975 bp DNA fragment containing extracellular domain 1-3 kdr, was designed according to the nucleotide sequence at GenBank and protein sequence at Swiss-Prot. The recombinant secretory expression vector (pPinkαHC/KDR1-3) was constructed and transferred into yeast by electroporation. The high expression transformants were identified through complementation of adenine auxotrophy and cultured. KDR1-3 was expressed under the induction of %1 methanol and confirmed by using SDS-PAGE and western blot techniques. After being purified by affinity chromatography using Ni-NTA resins, binding of expressed product to hVEGF165 was proved by two direct ELISA and ELISA receptor binding assays. The data showed that human VEGFR-2 extracellular domain 1-3 with eukaryotic protein structure, that there is no reported paper about, was successfully expressed. 
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Article Type: Original Research | Subject: Molecular biotechnology
Received: 2019/05/14 | Accepted: 2019/11/24 | Published: 2020/06/6

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