Cloning, expression, purification and bioactivity identification of murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in Escherichia coli

Abstract

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a cytokine that stimulates a variety of blood cells and therefore has many potential applications, such as chemotherapy side-effect reduction, bonemarrow cell immobilization, vaccine adjuvant and so on. Before GM-CSF can be used in human, it is required an initial trial on animal model. In this study, mGM-CSF was expressed in Escherichia coli (E. coli) to provide material for testing in mouse model. A gene mgm-csf coding for mGM-CSF protein was amplified from pIDTGM-CSF plasmid by PCR method, digested with restriction enzymes and inserted into pET28a plasmid to create a recombinant plasmid pET-mGM. The gene in the obtained plasmid was checked by PCR and sequencing. The plasmid was then transformed into the expressing strain E. coli BL21(DE3). After induction using IPTG, GM-CSF was over-expressed in the form of inclusion body in the cytoplasm of E. coli BL21(DE3) and was confirmed by Western blot with anti-GM-CSF antibodies. The inclusion bodies was then refolded and subjected for cation chromatography purification. The purified recombinant protein was mGMCSF with the purity up to 94.9 percent of total purified fraction. Finally, mGM-CSF bioactivity was assessed via the proliferation of FDC-P1. Result showed that the ED50 of mGM-CSF was 3022 pg/ml.