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1.  Expression and Purification of Functional Human Vascular Endothelial Growth Factor-A121; the Most Important Angiogenesis Factor 
Advanced Pharmaceutical Bulletin  2014;4(4):323-328.
Purpose: Angiogenesis or formation of new blood vessels is an essential process for tumor growth, invasion and metastasis. Vascular Endothelial Growth Factor (VEGF) and its receptors play an important role in angiogenesis-dependent tumors. VEGF-A is the most important factor in angiogenesis process. Human VEGF-A gene consists of eight exons that undergoes alternative exon splicing and produce five different proteins consisting of 121, 145, 165, 189 and 206 amino acids (named VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206).
Methods: In this study, VEGF121 gene synthesized and cloned into the pET-26b plasmid. The recombinant plasmid was transferred into appropriate expression strain of BL-21. Expression of VEGF121 induced by IPTG (Isopropyl β-D-1-thiogalactopyranoside) and confirmed by SDS-PAGE and Western-Blotting. Recombinant VEGF121 was purified by nickel affinity chromatography. HUVECs (Human Umbilical Vein Endothelia Cells) cells were isolated from umbilical vein and the effect of VEGF121 on tube formation of endothelial cells was investigated.
Results: SDS-PAGE and Western-Blotting results verified the purification of VEGF121. The final yield of recombinant protein was about 5mg per liter. Endothelial cell tube formation assay results showed that VEGF121 leads to tube formation of endothelial cell on matrix and induces angiogenesis in vitro.
Conclusion: Recombinant VEGF121 is important factor in tube formation of endothelial cell, so it could be used in different cancer researches and angiogenesis assay.
doi:10.5681/apb.2014.047
PMCID: PMC4137420  PMID: 25436186
VEGF121; Angiogenesis; Endothelial tube formation assay
2.  Expression of Recombinant Human Coagulation Factor VII by the Lizard Leishmania Expression System 
The variety of recombinant protein expression systems have been developed as a resource of FVII gene expression. In the current study, the authors used a novel protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host for expression of FVII. Plasmid containing cDNA encoding full-length human FVII was introduced into Lizard Leishmania and positive transfectants were analyzed by SDS-PAGE and Western blot analysis. Furthermore, biological activity of purified protein was detected by PT assay. The recombinant strain harboring a construct was analyzed for expression of FVII at the mRNA and protein level. Purified rFVII was obtained and in order to confirm the purified compound was in fact rFVII. Western blot analysis was carried out. Clotting time in PT assay was reduced about 30 seconds with the purified rFVII. In Conclusion, this study has demonstrated, for the first time, that Leishmania cells can be used as an expression system for producing recombinant FVII.
doi:10.1155/2011/873874
PMCID: PMC3168907  PMID: 21912483
3.  Purification and Refolding of Overexpressed Human Basic Fibroblast Growth Factor in Escherichia coli 
This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8 M urea. The adsorption was made in expanded bed without clarification steps such as centrifugation. Column refolding was done by elimination of urea and elution with NaCl. The human basic fibroblast growth factor was obtained as a highly purified soluble monomer form with similar behavior in circular dichroism and fluorescence spectroscopy as native protein. A total of 92.52% of the available human basic fibroblast growth factor was recovered as biologically active and purified protein using the mentioned purification and refolding process. This resulted in the first procedure describing high-throughput purification and refolding of human basic fibroblast growth factor in one step and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.
doi:10.4061/2011/973741
PMCID: PMC3151512  PMID: 21837279
4.  Chemical Assistance in Refolding of Bacterial Inclusion Bodies 
Escherichia coli is one of the most widely used hosts for the production of recombinant proteins but insoluble expression of heterologous proteins is a major bottleneck in production of recombinant proteins in E. coli. In vitro refolding of inclusion body into proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct folding. Cosolutes play a major role in refolding process and can be classified according to their function as aggregation suppressors and folding enhancers. This paper presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial processes.
doi:10.1155/2011/631607
PMCID: PMC3148444  PMID: 21822494

Results 1-4 (4)