|Production Design of Efficient Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor Under a Gene-Specific Promoter in Prokaryotic System|
|Mahmoud Vahidi1, Mojgan Bandehpour2,3, Bahram Kazemi2,3, Mahmood Bozorgmehr4|
|1Department of laboratory Science, Faculty of Paramedicine, AJA University of Medical Science, 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
CJMB 2020; 7: 314-321
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Keywords : Promoter, Escherichia coli, Recombinant human GM-CSF
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Objectives: Recombinant products play an important role in improving health conditions. In addition, human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is considered a cytokine which stimulates many differentiated myeloid cells in order to produce granulocytes, macrophages, and monocytes. Considering the clinical application of the human GM-CSF, the current study aimed to produce the recombinant human GM-CSF (rhGM-CSF) in the prokaryotic system and then evaluated its biological activity.
Materials and Methods: In this experimental study, the hGM-CSF was synthesized under a specific promoter. Then, it was cloned in HindIII restriction enzyme sites of the pcDNA3.1 (+). The hGM-CSF gene cloning was assessed by polymerase chain reaction, restriction enzyme digestion, and sequencing. Subsequently, recombinant plasmids were transformed in Escherichia coli and the expression of recombinant hGM-CSF was analyzed by electrophoresis and immunoblotting. Then, the rhGM-CSF was purified using S-tag affinity chromatography and the concentration of the purified rhGM-CSF was determined by ELISA. Finally, the biological function of the rhGM-CSF on TF-1 cells was performed by MTT proliferation assay.
Results: The cloned fragment on gel agarose was detected. Further, the restriction enzyme digestion and recombinant plasmid sequencing results confirmed pcDNA3.1 (+)/hGM-CSF cloning. Furthermore, the results of the expression analysis of rhGM-CSF by SDS-PAGE and western blot showed a specific protein band. The concentration of the purified protein was 0.54 μg/mL. Moreover, the proliferation index demonstrated that the treated cells were proliferated (P < 0.05). The mean values of the proliferation index were 7.8.
Conclusions: In general, the production of recombinant hGM-CSF protein in the prokaryotic system was simple, rapid, and inexpensive. Therefore, the functional rhGM-CSF can be expressed under gene-specific promoter without any need for the chemical inducer.
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