EXPRESSION OF cDNA FOR RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR IN ESCHERICHIA COLI AND CHARACTERIZATION OF THE PROTEIN
Abstract
Objective: To determine the biological activity of rhG-CSF and it's characterization.
Methods: The prokaryotic expression vector pG01 containing human GCSF cDNA were constructed with DNA recombination technology.
Results: We had achieved high level expression of the human G-CSF in E. coli, where it represented at least 23.6% of the total protein as determined from SDS-PAGE gels. The human G-CSF was expressed as inclusion bodies in E.coli. The inclusion bodies were soluhilized in a solution containing 7M urea, renatured by dialysis, isolated and purified by DEAEsepharose CL-6B ion exchange and Superdex 75 gel filtration chromatography. The purified rhG-CSF was confirmed by coincidence of biological activity and protein demonstrated by SDS-PAGE. It was homogeneous with respect to mol. Wt (18400). The purity of the rhGCSF might be >90 per cent.
Conclusion: The purified rhG-CSF in our laboratory had dramatically the biological activity of regulating proliferation and differentiation of the human G-CSF-dependent cell line NSF-I and the progenitor cells of granulocytes of human bone marrow.
Methods: The prokaryotic expression vector pG01 containing human GCSF cDNA were constructed with DNA recombination technology.
Results: We had achieved high level expression of the human G-CSF in E. coli, where it represented at least 23.6% of the total protein as determined from SDS-PAGE gels. The human G-CSF was expressed as inclusion bodies in E.coli. The inclusion bodies were soluhilized in a solution containing 7M urea, renatured by dialysis, isolated and purified by DEAEsepharose CL-6B ion exchange and Superdex 75 gel filtration chromatography. The purified rhG-CSF was confirmed by coincidence of biological activity and protein demonstrated by SDS-PAGE. It was homogeneous with respect to mol. Wt (18400). The purity of the rhGCSF might be >90 per cent.
Conclusion: The purified rhG-CSF in our laboratory had dramatically the biological activity of regulating proliferation and differentiation of the human G-CSF-dependent cell line NSF-I and the progenitor cells of granulocytes of human bone marrow.