Submitted: 17 Mar 2014
Accepted: 21 Apr 2014
ePublished: 25 Sep 2014
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Avicenna J Med Biochem. 2014;2(1): 5.
doi: 10.17795/ajmb-19002
  Abstract View: 1307
  PDF Download: 1475
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Research Article

Cloning and Expression of Human Keratinocyte Growth Factor in Escherichia coli for Recombinant Drug Production

Fatemeh Ebrahimzadeh 1, Yeganeh Talebkhan 2, Hassan Mirzahoseini 2, Ghasem Barati 1, Massoud Saidijam 1*

1 Research Center for Molecular Medicine, Department of Molecular Medicine and Genetics, Hamadan University of Medical Sciences, Hamadan, IR Iran
2 Department of Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, IR Iran
*Corresponding author: Massoud Saidijam, Research Center for Molecular Medicine, Department of Molecular Medicine and Genetics, School of Medicine, Hamadan University of Medical Sciences, Hamadan, IR Iran. Tel: +98-9121324616, Fax: +98-8138380464, Sjam110@yahoo.com


Background: Keratinocyte growth factor (KGF) is a member of fibroblast growth factor (FGF) family which induces proliferation and differentiation in a wide variety of epithelial tissues. KGF plays an important role in protection, repair of various types of epithelial cells, and re-epithelialization of wounds. Therefore, in patients with hematologic malignancies receiving high doses of chemotherapy and radiotherapy, treatment with KGF decreases the incidence and duration of severe oral mucositis.

Objectives: The aim of this study was to express the recombinant form of human keratinocyte growth factor in Escherichia coli.

Materials and Methods: KGF gene was amplified by PCR and cloned into the expression vector pET28a(+). The recombinant vectors were transformed into E. coli BL21(DE3) as expression host and expression of the desired protein was induced by IPTG. The expression was evaluated at RNA and protein levels by reverse transcriptase PCR (RT-PCR) and SDS-PAGE analyses, respectively and the expressed protein was confirmed through western blotting.

Results: Cloning was confirmed by PCR and restriction digestion. RT-PCR and SDS-PAGE represented expression of KGF in E. coli. The optimized expression was achieved 16 hours after induction with 0.3 mM IPTG at 37°C in luria broth (LB) containing kanamycin. The 18 kDa protein was confirmed by western blotting, using anti-His antibodies.

Conclusions: The result of the present study indicated that E. coli expression system was suitable for overexpression of recombinant human KGF and the expressed protein can be considered as a homemade product.

Keywords: Cloning, Recombinant Protein, Keratinocyte Growth Factor

Implication for health policy/practice/research/medical education:

In order to make a home-made recombinant human KGF, PET system and E. coli could be applied by Iranian researchers. As our knowledge, this is the first project in our country about this manner.

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