Overexpression of recombinant human granulocyte colony - stimulating factor in E. coli
IJMS-Iranian Journal of Medical Sciences. 2003; 28 (3): 131-4
in English
| IMEMR
| ID: emr-62287
ABSTRACT
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. 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]. 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. 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
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Index:
IMEMR (Eastern Mediterranean)
Main subject:
Escherichia coli
Language:
English
Journal:
Iran. J. Med. Sci.
Year:
2003
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