A Combined Refolding Technique for Recombinant Human Interferon-γ Inclusion Bodies by Ion-exchange Chromatography with a Urea Gradient |
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Authors: | Ting Jin Yi-Xin Guan Zheng-Zheng Fei Shan-Jing Yao Man-Gi Cho |
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Institution: | (1) Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou, 310027, P.R. China;(2) Engineering Research Center, Dongseo University, Busan, 616-010, Korea |
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Abstract: | Summary A refolding strategy was described for on-column refolding of recombinant human interferon-γ (rhIFN-γ) inclusion bodies by ion-exchange chromatography (IEC). The rhIFN-γ was expressed in E. colias inclusion bodies. Triton X-100 was used first to wash the rhIFN-γ inclusion bodies before chromatographic refolding. The refolding process was performed by gradually decreasing the concentration
of urea in the column after the denatured rhIFN-γ protein had bound onto the ion-exchange gel SP-Sepharose Fast Flow. The refolding and purification process for the denatured
rhIFN-γ was carried through simultaneously and the purity of the refolded rhIFN-γ was up to 95%. The effects of protein loading, flow rate, urea gradient length and final urea concentration on the refolding
were investigated in detail. Under the optimum conditions, the specific activity of rhIFN-γ was up to 7.5 × 105 IU mg−1and active protein recovery was up to 54%. |
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Keywords: | Gradient elution inclusion body ion-exchange chromatography recombinant human interferon-γ refolding |
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