Abstract
A simple fed-batch process was carried out using constant and variable specific growth rates for high-cell-density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-γ(hIFN-γ). The feeding rate was adjusted to achieve an appropriate specific growth rate. The dissolved oxygen level was maintained at 20–30% of air saturation by control of airflow and stirrer speed and, where necessary, by enrichment of inlet air with pure oxygen. Glucose was the sole source of carbon and energy and was provided by following a simple exponential feeding rate. The final cell density in the fed-batch fermentation with constant and variable specific growth rate feeding strategies was ~100 g dry cell wt l−1 after 36 and 20 h, respectively. The final specific yield and overall productivity of recombinant hIFN-γ in the variable specific growth rate strategy were 0.35 g rHu-IFN-γ g−1 dry cell wt and 0.9 g rHu-IFN-γ l−1 h−1, respectively. A new chromatographic purification procedure involving anion exchange and cation exchange chromatographies was developed for purification of rHu-IFN-γ from inclusion bodies. The established purification process is reproducible and the total recovery of rHu-IFN-γ was ~30% (100 mg rHu-IFN-γ g−1 dry cell wt). The purity of the rHu-IFN-γ was determined using HPLC. Sterility, pyrogenicity, and DNA content tests were conducted to assure the absence of toxic materials and other components of E. coli in the final product. The final purified rHu-IFN-γ has a specific antiviral activity of ~2×107 IU/mg protein, as determined by viral cytopathic effect assay. These results certify the product for clinical purposes.
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The authors acknowledge support for part of this research by the Noor Research and Educational Institute and the Shafa-e-Sari Antibiotic Producing Company, Iran.
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Khalilzadeh, R., Shojaosadati, S.A., Maghsoudi, N. et al. Process development for production of recombinant human interferon-γ expressed in Escherichia coli . J IND MICROBIOL BIOTECHNOL 31, 63–69 (2004). https://doi.org/10.1007/s10295-004-0117-x
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DOI: https://doi.org/10.1007/s10295-004-0117-x