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Optimization of conditions for expression of recombinant interferon-γ in E.coli

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Abstract

Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli.

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  1. Defence Institute of Physiology and Allied Sciences, Defence Research and Development Organization, New Delhi, 110054, India

    Meetul Kumar, Mrinalini Singh & Shashi Bala Singh

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  1. Meetul Kumar
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  2. Mrinalini Singh
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  3. Shashi Bala Singh
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Correspondence to Meetul Kumar.

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Kumar, M., Singh, M. & Singh, S.B. Optimization of conditions for expression of recombinant interferon-γ in E.coli . Mol Biol Rep 41, 6537–6543 (2014). https://doi.org/10.1007/s11033-014-3537-3

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  • DOI: https://doi.org/10.1007/s11033-014-3537-3

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