Simplified Large-Scale Refolding,Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli
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| Authors: | Chang Kyu Kim Chi Ho Lee Seung-Bae Lee Jae-Wook Oh |
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| Institution: | 1. Department of Animal Biotechnology/Animal Resources Research Center, College of Animal Bioscience and Biotechnology, Konkuk University, Seoul, Korea.; 2. Department of Food Science and Biotechnology of Animal Resources, College of Animal Bioscience and Biotechnology, Konkuk University, Seoul, Korea.; 3. Division of Animal Resources and Life Science, Sangji University, Wonju, Korea.; Tecnologico de Monterrey, Mexico, |
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| Abstract: | 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) 2nd 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. |
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