Cloning of human granulocyte-macrophage colony stimulating factor (GM-CSF) cDNA and its expression in E-coli

Human GM-CSF cDNA fragment encoding the mature GM-CSF was obtained by using RT-FCR method with total RNA extracted from induced human fetal lung cells HFL. The sequence of the hGM-CSF cDNA thus obtained was the same as those reported. In order Go get expression of a high level in E. coli, the 5' terminal nucleotide sequence of hGM-CSF cDNA was modified by using PCR. the modified hGM-CSF cDNA was inserted into plasmid pET-11d containing T7 promoter, with the resulted expression of plasmid pETC-5. E. coli BL21 (DES) was transformed with pETC-5 and an expressed strain BLEC4 was selected. SDS-PAGE analysis revealed that rhGM-CSF was produced and accumunlated up to 16% of the total cellular protein in the form of Inclusion body in BLEC4 cells after induced by 0.5 mM IPTG for 2 h. ELISA and TF-I cell culture assay showed that the biological activity of the partially purified and renatured rhGM-CSF was similar to that of the natural human GM-CSF.