|Production Design of Efficient Recombinant Human GM-CSF (rhGM-CSF) Under a Gene-specific Promoter in Prokaryotic System|
|Mahmoud Vahidi1, Mojgan Bandehpour2,3, Bahram Kazemi2,3, Mahmood Bozorgmehr4|
|1Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran
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Keywords : Promoter, Escherichia coli, Recombinant human GM-CSF
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Objectives: Recombinant products play an important role to improve health conditions.Human Granulocyte-Macrophage Colony Stimulating Factor (hGM-CSF) is a cytokine that stimulates many differentiated myeloid cells to produce granulocytes, macrophages, and monocytes. Considering the clinical application of human GM-CSF, the current study is aimed to produce the recombinant human GM-CSF (rhGM-CSF) in Prokaryotic System and evaluated its biological activity.
Materials and Methods: In this experimental study the hGM-CSF under specific promoter was synthesized. Then it was cloned in HindIII restriction enzyme sites of the pcDNA3.1(+). The hGM-CSF gene Cloning was assessed by PCR, restriction enzyme digestion and sequencing. Subsequently, recombinant plasmids were transformed in Escherichia coli and expression of recombinant hGM-CSF was analyzed by electrophoresis and Immunoblotting. The rhGM-CSF purification was carried out by S-tag affinity chromatography and concentration of purified rhGM-CSF was determined by ELISA. Thebiological function of the rhGM-CSF on TF-1 cells was carried out by MTT proliferation assay.
Results: The cloned fragment on gel agarose was detected. Restriction enzyme digestion and recombinant plasmid sequencing results were confirmed pcDNA3.1(+)/hGM-CSF cloning. Expression analysis of rhGM-CSF by SDS-PAGE and Western blot showed a specific protein band. The concentration of purified protein was 0.54 μg/ml. Proliferation Index (PI) showed that treated cells were proliferated (p<0.05). The mean values of Proliferation Index were 7.8.
Conclusion: The production of recombinant hGM-CSF protein in the prokaryotic system was simple, rapid and inexpensive. We could express functional rhGM-CSF under gene-specific promoter no need to the chemical inducer.
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