Simplified large-scale refolding, purification, and characterization of recombinant human granulocyte-colony stimulating factor in Escherichia coli.
Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2(nd) International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins.
Department of Animal Biotechnology/Animal Resources Research Center, College of Animal Bioscience and Biotechnology, Konkuk University, Seoul, Korea.
Chang Kyu
CK
Kim
Chi Ho
CH
Lee
Seung-Bae
SB
Lee
Jae-Wook
JW
Oh
D004926:Escherichia coli / Q000378:metabolism*; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor