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[Modification of N-terminal cDNA of hGM-CSF and high-expression in E. coli]
1Department of Virology, Institute of Microbiology, Academia Sinica, Beijing 100080.
Objective:
N-terminal cDNA of human granulococyte-macrophage colony stimulating factor(GM-CSF) was designed to be modified and highly-expressed in E. coli.
Methods:
A pair of oligo-nucleotide primers were used to modify the mature N-terminal cDNA sequence of hGM-CSF with the method of PCR. the modified cDNA of hGM-CSF was cloned into E. coli. expressive vector PBV220 and expressed in E. coli DH5 alpha strain, the biological activities of the recombinant protein was identified by means of cell colony formation and TF-1 cell growth assay in vitro.
Results:
The expression level of modified hGM-CSF cDNA was higher than that of unmodified native type. SDS-PAGE revealed that expressed protein of hGM-CSF accounted for about 25% of total bacterial cell protein. The biological activity of recombinant protein was about 1.5 x 10(7) U/mg protein. The sequence of 1-16 amino acid of N-terminal of the recombinant protein was same with native hGM-CSF.
Conclusions:
Modification of the N-terminal cDNA of hGM-CSF could dramatically enhance the expression level in E. coli system.
