Human granulocyte colony stimulating factor (hG-CSF): cloning and expression in E. coli cells

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.