Heterologous expression of a truncated form of human recombinant vascular endothelial growth factor-A and its biological activity in wound healing

Document Type : Original Article


1 Molecular and Medicine Research Center, Department of Immunology and Microbiology, School of Medicine, Arak University of Medical Sciences, Arak, Iran

2 National Institute for Genetic Engineering and Biotechnology, Tehran, Iran

3 Department of Anatomy, School of Medicine, Arak University of Medical Sciences, Arak. Iran


Objective(s): Vascular endothelial growth factor (VEGF) is one of the most effective proteins in angiogenesis, mesenchymal stem cells (MSCs) differentiation and wound healing. These abilities are therapeutic potential of VEGF in diabetic retinopathy, nephropathy and other tissue damage circumstances. In this study, recombinant VEGF was produced in Escherichia coli (E. coli) system and then biological activity of this protein was evaluated in animal wound healing.
Materials and Methods: E. coli BL21 (DE3) competent cells were transformed with pET32a-VEGF clone and induced by isopropyl-β-D-thio-galactoside (IPTG). The recombinant protein was purified byaffinity chromatography. Recombinant VEGF-A-based ointment (VEGF/Vaseline 0.8 mg/100 w/w) was used for external wound (25×15mm thickness) healing in animal model. In vivo activity of ointment was evaluated by clinical evidences and cytological microscopic assessment.
Results: The recombinant protein with molecular weight of 45 kilodaltons (kDa) and concentration of 0.8 mg/ml was produced.Immunoblotting data showed that the antigenic region of VEGF can be expressed in E. coli and the recombinant protein has similar epitopes with close antigenic properties to the natural form. Macroscopic findings and microscopic data showed that the recombinant VEGF-A ointment was effective on excisional wound healing.
Conclusion: Recombinant VEGF-A produced by pET32a in E. coli, possesses acceptable structure and has wound healing capability.


1. Vempati P, Popel AS, Mac Gabhann F. Extracellular regulation of VEGF: isoforms, proteolysis, and vascular patterning. Cytokine Growth Factor Rev 2014; 25:1-19.
2. Lee SB, Park JS, Lee S, Park J, Yu S, Kim H, et al. Overproduction of recombinant human VEGF (vascular endothelial growth factor) in Chinese hamster ovary cells. J Microbiol Biotechnol 2008; 18:183-187.
3. Berendsen AD, Olsen BR. How vascular endothelial growth factor-A (VEGF) regulates differentiation of mesenchymal stem cells. J Histochem Cytochem 2014; 62:103-108.
4. Cai Q, Brissova M, Reinert RB, Pan FC, Brahmachary P, Jeansson M, et al. Enhanced expression of VEGF-A in beta cells increases endothelial cell number but impairs islet morphogenesis and beta cell proliferation. Dev Biol 2012; 367:40-54.
5. Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 1996; 60:512-538.
6. Hiley CT, Chard LS, Gangeswaran R, Tysome JR, Briat A, Lemoine NR, et al. Vascular endothelial growth factor A promotes vaccinia virus entry into host cells via activation of the Akt pathway. J Virol 2013; 87:2781-2790.
7. Igarashi Y, Chosa N, Sawada S, Kondo H, Yaegashi T, Ishisaki A. VEGF-C and TGF-beta reciprocally regulate mesenchymal stem cell commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes. Int J Mol Med 2016; 37:1005-1013.
8. Li C, Zhen G, Chai Y, Xie L, Crane JL, Farber E, et al. RhoA determines lineage fate of mesenchymal stem cells by modulating CTGF-VEGF complex in extracellular matrix. Nat Commun 2016; 7:11455.
9. Ikhapoh IA, Pelham CJ, Agrawal DK. Atherogenic cytokines regulate VEGF-A-induced differentiation of bone marrow-derived mesenchymal stem cells into endothelial cells. Stem Cells Int 2015; 2015:498328.
10. Fernandez-Alonso R, Martin-Lopez M, Gonzalez-Cano L, Garcia S, Castrillo F, Diez-Prieto I, et al. p73 is required for endothelial cell differentiation, migration and the formation of vascular networks regulating VEGF and TGFbeta signaling. Cell Death Differ 2015; 22:1287-1299.
11. Sorensen HP, Mortensen KK. Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 2005; 115:113-128.
12. Seyedarabi A, Cheng L, Zachary I, Djordjevic S. Production of soluble human vascular endothelial growth factor VEGF-A165-heparin binding domain in Escherichia coli. PLoS One 2013; 8:e55690.
13. Moghadam M, Ganji A, Varasteh A, Falak R, Sankian M. Refolding process of cysteine-rich proteins:Chitinase as a model. Rep Biochem Mol Biol 2015; 4:19-24.
14. Legeza VI, Grebenyuk AN, Kondakov AY, Zargarova NI. [Comparative evaluation of healing wounds at a local and combined radiation injury in an experiment]. Radiats Biol Radioecol 2015; 55:584-590.
15. Galiano RD, Michaels Jt, Dobryansky M, Levine JP, Gurtner GC. Quantitative and reproducible murine model of excisional wound healing. Wound Repair Regen 2004; 12:485-492.
16. Stuart DA, Oorschot DE. Embedding, sectioning, immunocytochemical and stereological methods that optimise research on the lesioned adult rat spinal cord. J Neurosci Methods 1995; 61:5-14.
17. Sambrook J, Russell DW. Molecular cloning: a laboratory manual 3rd edition. UK: Coldspring-Harbour Laboratory Press; 2001.
18. Molaee N, Abtahi H, Mosayebi G. Expression of recombinant streptokinase from streptococcus pyogenes and its reaction with infected human and murine sera. Iran J Basic Med Sci 2013; 16:985-989.
19. Sadoogh Abbasian S, Ghaznavi Rad E, Akbari N, Zolfaghari MR, Pakzad I, Abtahi H. Overexpression and enzymatic assessment of antigenic fragments of hyaluronidase recombinant protein from streptococcus pyogenes. Jundishapur J Microbiol 2015; 8:e13653.
20. Claffey KP, Senger DR, Spiegelman BM. Structural requirements for dimerization, glycosylation, secretion, and biological function of VPF/VEGF. Biochim Biophys Acta 1995; 1246:1-9.
21. Sugimura K, Higashi N. A novel outer-membrane-associated protease in Escherichia coli. J Bacteriol 1988; 170:3650-3654.
22. Abtahi H, Salmanian AH, Rafati S, Behzadian Nejad G, Mohammad Hassan Z. High level expression of recombinant ribosomal protein (L7/L12) from Brucella abortus and its reaction with infected human sera. Iran Biomed J 2004; 8:13-18.
23. Pizarro SA, Gunson J, Field MJ, Dinges R, Khoo S, Dalal M, et al. High-yield expression of human vascular endothelial growth factor VEGF(165) in Escherichia coli and purification for therapeutic applications. Protein Expr Purif 2010; 72:184-193.
24. Mendes JJ, Leandro CI, Bonaparte DP, Pinto AL. A rat model of diabetic wound infection for the evaluation of topical antimicrobial therapies. Comp Med 2012; 62:37-48.
25. Scrofani SD, Fabri LJ, Xu P, Maccarone P, Nash AD. Purification and refolding of vascular endothelial growth factor-B. Protein Sci 2000; 9:2018-2025.
26. Keswani SG, Balaji S, Le LD, Leung A, Parvadia JK, Frischer J, et al. Role of salivary vascular endothelial growth factor (VEGF) in palatal mucosal wound healing. Wound Repair Regen 2013; 21:554-562.
27. Nauta A, Seidel C, Deveza L, Montoro D, Grova M, Ko SH, et al. Adipose-derived stromal cells overexpressing vascular endothelial growth factor accelerate mouse excisional wound healing. Mol Ther 2013; 21:445-455.
28. Balaji S, LeSaint M, Bhattacharya SS, Moles C, Dhamija Y, Kidd M, et al. Adenoviral-mediated gene transfer of insulin-like growth factor 1 enhances wound healing and induces angiogenesis. J Surg Res 2014; 190:367-377.
29. Basso FG, Pansani TN, Turrioni AP, Bagnato VS, Hebling J, de Souza Costa CA. In vitro wound healing improvement by low-level laser therapy application in cultured gingival fibroblasts. Int J Dent 2012; 2012:719452.
30. Kusumanto YH, van Weel V, Mulder NH, Smit AJ, van den Dungen JJ, Hooymans JM, et al. Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: a double-blind randomized trial. Hum Gene Ther 2006; 17:683-691.