Overexpression of Recombinant Human Granulocyte Colony-Stimulating Factor in E. coli

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.