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Cloning, Expression, and Assessment of Cytotoxic Effects of A-Ngr Fusion Protein Publisher



Mohammadifarsani A1 ; Jahaniannajafabadi A2 ; Habibiroudkenar M3 ; Golkar M4 ; Shokrgozar MA5 ; Khanahmad H6 ; Golshani M1 ; Valiyari S1 ; Bouzari S1
Authors
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Authors Affiliations
  1. 1. Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran
  2. 2. Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
  3. 3. Medical Biotechnology Research Center, Paramedicine Faculty, Guilan University of Medical Sciences, Rasht, Iran
  4. 4. Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran
  5. 5. National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
  6. 6. Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

Source: International Journal of Peptide Research and Therapeutics Published:2018


Abstract

Targeted drug delivery is an attractive field in cancer studies. In this study, a novel fusion protein consisting of Shiga toxin A subunit and NGR peptide has been constructed. The cytotoxic Shiga toxin A subunit has the ability to kill cancer cells while NGR is a well-known peptide that targets the whole molecule to cancer cells. Two forms of this novel fusion protein, one without linker (A-NGR) and one with linker (A-GGGGS-NGR) were studied. 3D structure prediction of the two forms carried out by I-TASSER and their validation and analysis were performed by ProSA web and RAMPAGE. Results showed that A-NGR is a better model than the one with linker. A-NGR was constructed by PCR method and cloned in pBAD/gIII A vector. Then, it was successfully expressed in Escherichia coli by induction with arabinose and subsequently purified by affinity chromatography under denaturing condition. Ultimately, the cytotoxic effect of the purified protein was evaluated on U937 cancer cells and MRC-5 normal cells by MTT assay. Conclusively, the fusion protein was successfully cloned and expressed and evaluated for its cytotoxic effects. The IC50 value of A-NGR fusion protein for U937 cell was about 26.86 µg/ml while no cytotoxic effect was observed on MRC-5 cells. Therefore, considering the promising cytotoxic effects of the fusion protein, further in vitro evaluations of this fusion protein on different cell lines are underway. © 2017, Springer Science+Business Media, LLC.
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