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-PCR method with total RNA extracted from induced human fetal lung cells HFL. The sequence of the h GM-CSF cDNA thus obtained was the same as those reported. In order to get expression of a high level in, the 5' terminal nucleotide sequence of hGM-CSF cDNA was modified by using PCR. The modified h GM-CSF cDNA was inserted into plasmid pET-11d containing T7 promoter, with the resulted expression of plasmid pETC-5. E. coli BL21(DE3) was transformed with pETC-5 and an expressed strain BLEC4 was selected. SDS-PAGE analysis revealed the rhGM-CSF was produced and accumulated up to 16% of the total cellular protein in the form of inclusion body in BLEC$ cells after induced by 0.5 mM IPTG for 2 h. ELISA and TF-1 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.
Yao
Jun
J
Shanghai Institute of Biochemistry, Academia Sinica, Shanghai 200031, China.
Gan
Ren-Bao
RB
Zhang
Qian
Q
Li
Zai-Ping
ZP