Discovery of Leptulipin, a New Anticancer Protein from theIranian Scorpion, Hemiscorpius lepturus.

Cancer is one of the leading causes of mortality in the world. Unfortunately, the present anticancer chemotherapeutics display high cytotoxicity. Accordingly, the discovery of new anticancer agents with lower side effects is highly necessitated. This study aimed to discover an anticancer compound from Hemiscorpius lepturus scorpion venom. Bioactivity-guided chromatography was performed to isolate an active compound against colon and breast cancer cell lines. 2D electrophoresis and MALDI-TOF were performed to identify the molecule. A partial protein sequence was obtained by mass spectrometry, while the full-length was deciphered using a cDNA library of the venom gland by bioinformatics analyses and was designated as leptulipin. The gene was cloned in pET-26b, expressed, and purified. The anticancer effect and mechanism action of leptulipin were evaluated by MTT, apoptosis, and cell cycle assays, as well as by gene expression analysis of apoptosis-related genes. The treated cells displayed inhibition of cell proliferation, altered morphology, DNA fragmentation, and cell cycle arrest. Furthermore, the treated cells showed a decrease in BCL-2 expression and an increase in Bax and Caspase 9 genes. In this study, we discovered a new anticancer protein from H. lepturus scorpion venom. Leptulipin showed significant anticancer activity against breast and colon cancer cell lines.

In silico designing of a new cysteine analogue of hirudin variant 3 for site specific PEGylation.

Hirudin is an anticoagulant agent of the salivary glands of the medicinal leech. Recombinant hirudin (r-Hir) displays certain drawbacks including bleeding and immunogenicity. To solve these problems, cysteine-specific PEGylation has been proposed as a successful technique. However, proper selection of the appropriate cysteine residue for substitution is a critical step. This study has, for the first time, used a computational approach aimed at identifying a single potential PEGylation site for replacement by cysteine residue in the hirudin variant 3 (HV3). Homology modeling (HM) was performed using MODELLER. All non-cysteine residues of the HV3 were replaced with the cysteine. The best model was selected based on the results of discrete optimized protein energy score, PROCHECK software, and Verify3D. The receptor binding was investigated using protein-protein docking by ClusPro web tool which was then visualized using LigPlot+ software and PyMOL. Finally, multiple sequence alignment (MSA) using ClustalW software and disulfide bond prediction were performed. According to the results of HM and docking, Q33C, which was located on the surface of the protein, was the best site for PEGylation. Furthermore, MSA showed that Q33 was not a conserved residue and LigPlot+ software showed that it is not involved in the hirudin-thrombin binding pocket. Moreover, prediction softwares established that it is not involved in disulfide bond formation. In this study, for the first time, the utility of the in silico approach for creating a cysteine analogue of HV3 was introduced. Our study demonstrated that the substitution of Q33 by cysteine probably has no effect on the biological activity of the HV3. However, experimental analyses are required to confirm the results.

Inhibition of angiogenesis in human endothelial cell using VEGF specific nanobody.

Angiogenesis is an important step in tumor development and metastasis. Vascular endothelial growth factor (VEGF) plays an important role in progression of angiogenesis. VEGF121 and VEGF165 are the most relative forms of VEGF family which contain the full biological activity. Nanobodies derived from camelidae are the smallest biding site of antigen. Unique characteristic of nanobodies make them as a useful candidate for research. In this report, we describe the isolation of VEGF specific nanobodies from dromedaries immunized with purified VEGF antigen using phage display. Four clones that showed the highest signal value in ELISA experiment were selected and expressed as a His-tagged fusion protein. Four selected nanobodies were reacted strongly to VEGF in cross-reactivity assay. The binding affinity of selected nanobodies named Nb22, Nb23, Nb35 and Nb42 were differed from 0.1 to 60nM. The nanobodies inhibited endothelial cell proliferation or tube formation in response of VEGF in a dose-dependent manner. These results indicate the potential of nanobodies in inhibition of VEGF and represent a promising candidate for cancer research and therapeutics.

Expression and purification of functional human vascular endothelial growth factor-a121; the most important angiogenesis factor.

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 ß-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.

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.