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Expression and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor in Fed-Batch Culture of Escherichia coli

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Abstract

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that has multiple roles in hematopoietic cells such as the regulation of proliferation and differentiation. Here, we describe fed-batch culture, refolding, and purification of rhG-CSF. The suitability of urea or sarcosine for solubilizing inclusion bodies (IBs) was tested. It was observed that urea is more efficient for solubilizing and refolding IBs than sarcosine is. The purity of rhG-CSF and the removal percentage of the rhG-CSF isoforms during purification were increased by pH 5.5 precipitation. The purity and the yield of purified rhG-CSF were 99 % and 0.5 g of protein per liter culture broth, respectively. Our protocols of recombinant protein purification using ion exchange chromatography and semipreparative high performance liquid chromatography of pH-precipitated refolded solution may be informative to the industrial scale production of biopharmaceuticals.

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Acknowledgments

This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (NRF-2008-313-E00238).

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Authors and Affiliations

  1. Department of Animal Biotechnology/Animal Resources Research Center, College of Animal Bioscience and Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin gu, Seoul, 143-701, South Korea

    Chang-Kyu Kim, Jun-Ha Choi & Jae-Wook Oh

  2. Division of Animal Resources and Life Science, Sangji University, Wonju, 220-702, South Korea

    Seung-Bae Lee

  3. Department of Life Science, Cheongju University, Cheongju, Chungbuk, 360-764, South Korea

    Sang-Mahn Lee

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  1. Chang-Kyu Kim
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  2. Jun-Ha Choi
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  3. Seung-Bae Lee
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Correspondence to Jae-Wook Oh.

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Supplemental Figure 1

IEF analyses of rhG-CSF eluted by SP-sepharose chromatography SP-sepharose chromatography was performed to purify rhG-CSF protein. Each fraction obtained was analyzed by Novex® pH 3–10 IEF system. rhG-CSF samples were employed to examine the purity. IEF markers indicate PI. Lane 1, pI marker 4.5–7.4. (PPTX 73.9 kb)

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Kim, CK., Choi, JH., Lee, SB. et al. Expression and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor in Fed-Batch Culture of Escherichia coli . Appl Biochem Biotechnol 172, 2425–2435 (2014). https://doi.org/10.1007/s12010-013-0708-y

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  • DOI: https://doi.org/10.1007/s12010-013-0708-y

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