Production and on-column re-folding of human vascular endothelial growth factor 165 in Escherichia coli

• Published in: Biotechnology and bioprocess engineering Vol. 18; no. 5; pp. 835 - 842
• Main Authors: Bang, Sun Kwon, Kim, Young Sik, Chang, Byung Soo, Park, Cheol Beom, Bang, In Seok
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.09.2013; Springer Nature B.V; 한국생물공학회
• Subjects: Angiogenesis; Biotechnology; Chemistry; Chemistry and Materials Science; Chromatography; Cloning; E coli; Escherichia coli; Gene expression; Industrial and Production Engineering; Kinases; Physiology; Protein folding; Proteins; Reagents; Research Paper; Studies; Vascular endothelial growth factor; 생물공학; Korea; human VEGF; protein refolding; angiogenesis; bacterial expression system
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-012-0829-0

Vascular endothelial growth factors (VEGFs) are a family of proteins that promote angiogenesis and participate in a variety of physiological and pathological processes. In this work, the gene encoding the human VEGF isoform 165 (hVEGF 165 ) was cloned into the expression vector pET32a (+) to construct a fusion expression plasmid that induced the thioredoxin (Trx) gene and transformed into Escherichia coli . The recombinant fusion protein TrxhVEGF 165 was expressed optimally as inclusion bodies in the case of being cultivated for 4 h at 30°C and 1 mM IPTG concentration. The Trx-hVEGF 165 was refolded and purified effectively from urea-solubilized inclusion bodies by the immobilized metal affinity chromatography. Released from the fusion protein by enterokinase cleavage and purified to homogeneity, the recombinant hVEGF 165 (rhVEGF 165 ) was biologically active as assessed by the human umbilicalvein endothelial cells (HUVECs) proliferation and the chicken chorioallantoic membrane (CAM) assay. The expression and in vitro refolding of rhVEGF 165 resulted in production of an active molecule in a yield of 4.04 mg/L flask cultivation.