Human granulocyte colony stimulating factor (hG-CSF): cloning and expression in E. coli cells | ||||
| Arab Journal of Biotechnology | ||||
| Article 4, Volume 18, Issue 1, June 2015, Page 45-54 PDF (524.01 K) | ||||
| Document Type: Original Article | ||||
| DOI: 10.21608/arjb.2015.428410 | ||||
 View on SCiNiTO | ||||
| Abstract | ||||
| The human granulocyte colony-stimulating factor (hG-CSF) is a growth factor or cytokine produced by a number of different tissues to stimulate the bone marrow for granulocytes and stem cells production. In this study, the hG-CSF mRNA was isolated from human peripheral blood mononuclear cells (PBMCs) cultured in-vitro and induced by lipo-polysaccharide. The cDNA of hG-CSF was amplified by PCR followed by DNA sequencing. Sequence analysis using NCBI Blast program revealed a 100% homology to the human G-CSF. The hG-CSF cDNA fragment was inserted into pET-3a expression vector and transformed into TOP-10 competent E. coli cells. The recombinant pET-3a/hG-CSF plasmid was transformed into different strains of BL21 E. coli cells. It was successfully expressed in BL21-Gold (DE3) strain. Western blotting of recombinant bacterial lysate using anti-human G-CSF showed a positive band at the MW of ~19 kDa. This confirms that the expressed protein has the same molecular weight and the immunogenicity against the specific antibodies of hG-CSF. Results proved that the E. coli strain BL21-Gold (DE3) carrying the construct pET-3a/hG-CSF is expressing rhG-CSF protein efficiently and can be used for scaling-up production. | ||||
| Keywords | ||||
| Granulocyte colony-stimulating factor (G-CSF); Growth factor; pET-3a expression system; Cloning; Gene expression | ||||
|
Statistics Article View: 35 PDF Download: 10 |
||||
