Cloning of Human Granulocyte-macrophage Colony Stimulating Factor (GM-CSF) cDNA and Its Expression in E. coli.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai)
; 28(3): 265-271, 1996.
Article
in En
| MEDLINE
| ID: mdl-12237681
ABSTRACT
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.
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Collection:
01-internacional
Database:
MEDLINE
Language:
En
Journal:
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai)
Year:
1996
Document type:
Article
Affiliation country:
China