Document Type : Original Article(s)
Authors
- M J Fallah 1
- B Akbari 1
- A.R Saeedinia 1
- M Karimi 2
- M Vaez 1
- M Zeinoddini 1
- M Soleimani 3
- N Maghsoudi 1
1 Department of Genetic Engineering, Research Center for Science and Biotechnology, Tehran, Iran
2 Department of Biotechnology, Pasteur Institute of Iran, Tehran, Iran
3 Department of Hematology, Tarbiat Modarres University, School of Medicine,Tehran, Iran
Abstract
Bakground: Granulocyte colony-stimulating factor (G-CSF) is a cytokine that stimulates hematopoiesis and induces proliferation and differentiation of granulocyte progenitor cells as well as production of bone marrow neutrophilic granulocyte colonies. Nowadays, human recombinant G-CSF(hr G-CSF)is used for the treatment of chemotherapy- and radiotherapy-induced neutropenia, and also in patients with bone marrow transplantation. Methods: A cDNA of human G-CSF (hG-CSF) was synthesized by PCR from recombinant cloning vector, with two altered nucleotides for increasing mRNA stability and overexpression, then inserted into a pET expression vector under the control of T7 promoter and cloned in E. coli strain BL21 (DE3). Results: After culture and induction of recombinant E. coli with IPTG, we achieved a high level expression of the hG-CSF, where it represented approximately 35% of the total protein as determined by SDS-PAGE and confirmed by western blotting with polyclonal and monoclonal hG-CSF antibodies. Conclusion: rhG-CSF was produced in a significantly high quantity with a yield of 35% of total protein as determined by SDS-PAGE. Since it is easily obtained by simple purification steps, it may be cost-effective, even on an industrial scale.
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