Extended Abstract
Introduction
Vascular Endothelial Growth Factor (VEGF) is a glycoprotein that is produced in various cells including macrophages, platelets, keratinocytes, renal mesangial cells as well as a variety of cancer cells. VEGF has angiogenic and mitogenic roles, differentiating cells, enhancing angiogenesis and repairing tissues. The most common and most important subset of this protein is VEGF-A-165 [3]. Production of recombinant proteins including VEGF in prokaryotic cells, impair their function due to disruption of protein structure. Since the process of modifying the protein structure by conventional methods such as chemical dialysis is time consuming and expensive, in the present study, appropriate additives were selected for structural modification and restoration of the activity of VEGF, and then these additives were used for chemical dialysis of the recombinant VEGF in vitro.
Materials and Methods
In this experimental in vitro study, the gene encoding VEGF-A165 (Acc: NM_001287044), was extracted from NCBI database. By adding the sequence of BamHI and XhoI restriction enzymes to the gene, the fragment was synthesized by the Biomatics Company. After transformation of the gene into E. coli DH5α proliferative cells and E. coli BL21 (DE3) expression cells, protein expression induction was performed with IPTG [7-9]. Recombinant protein was extracted by affinity chromatography and Ni-NTA kit. The presence of protein was confirmed by SDS-PAGE method. To simulate the structural modification process of proteinase, ExPASy server, aggrescan server, PDB, Chimera Photo, PubChem, Hyperchem, AutoDock and LigPlot software were used. The selected additives used were used in these nine dialysis programs and the product of each program was evaluated by flow cytometry for treatment of mesenchymal stem cell (MSc) and its differentiation into endothelial cell (EC). Commercial protein (ab9571, Abcam Co.) was used as positive control. Data were analyzed by independent T-test and Mann-Whitney U test in SPSS software considering a significance level of less than 0.05.
Results
The results of the LigPlot software showed that weaker hydrogen bonds were formed between cysteine and VEGF compared to other amino acids. The aggrescan server data showed sensitive areas of protein aggregation. Based on flow cytometry results, the rate of specific cluster differentiation markers (CD31/CD144) in the recombinant VEGF-treated group was 27%; in the commercial protein-treated group, 17%; and in the control group, 15%.
Discussion
The greater impact of recombinant VEGF than commercial protein on cell differentiation reported in this study may be due to the protein structure modification by using software and using appropriate chemical additives for chemical dialysis of this protein. According to the results of this study, cysteine had the most effect on the structural modification of recombinant VEGF. This result was consistent with the software results because the level of bonding energy between this amino acid and VEGF was lower and the hydrogen bonds between them were higher than the others. Cysteine can facilitate cross-linking of the disulfide bonds in the structure of recombinant VEGF. The effect of cysteine along with dithiothreitol (DTT) on modifying the structure of the recombinant VEGF is remarkable because DDT acts as a redox compound of the common disulfide bonds, which occurs more frequently under alkaline buffer conditions. Ethylenediaminetetraacetic Acid (EDTA), arginine and triton X-100 also had a reinforcing role in modifying the structure of recombinant VEGF. EDTA is a chelator and inhibitor of metalloprotease enzymes and reduces oxidation reactions and enhances protein solubility. Triton X-100 is a non-ionic surfactant and a lubricant detergent; it stops protein aggregation and by inducing the solubility of the oligomeric proteins, supports the refolding process.
Ethical Considerations
Compliance with ethical guidelines
This study obtained its Ethical approval from the Research Ethics Committee of Arak University of Medical Sciences (Code: ARAKMU. REC.1394.199). All experiments in this study on living cells in standard laboratory conditions, were carried out in compliance with the principles of biosafety.
Funding
This study is a research proposal approved by Arak University of Medical Sciences (code: 2356) and received financial support from the Deputy for Research and Technology.
Authors' contributions
Scientific design and management: Hamid Abtahi; Design and implementation of flow cytometry: Ghasem Mosibi; Implementation of practical research process and writing an article: Mohsen Khaki.
Conflicts of interest
The authors would like to thank the Deputy for Research and Technology of Arak University of Medical Sciences for their valuable support.