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Optimization of Human Granulocyte Macrophage-Colony Stimulating Factor (hGM-CSF) Expression Using Asparaginase and Xylanase Gene’s Signal Sequences in Escherichia coli

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Abstract

The toxicity of the recombinant protein towards the expression host remains a significant deterrent for bioprocess development. In this study, the expression of human granulocyte macrophage-colony stimulating factor (hGM-CSF), which is known to be toxic to its host, was enhanced many folds using a combination of genetic and bioprocess strategies in Escherichia coli. The N terminus attachment of endoxylanase and asparaginase signal sequences from Bacillus subtilis and E. coli, respectively, in combination with and without His-tag, considerably improved expression levels. Induction and media optimization studies in shake flask cultures resulted in a maximal hGM-CSF concentration of 365 mg/L in the form of inclusion bodies (IBs) with a specific product yield (Y P/X) of 120 mg/g dry cell weight in case of the asparaginase signal. Culturing the cells in nutrient rich Terrific broth maintained the specific product yields (Y P/X) while a 6.6-fold higher volumetric concentration of both product and biomass was obtained. The purification and refolding steps were optimized resulting in a 95% pure protein with a fairly high refolding yield of 45%. The biological activity of the refolded protein was confirmed by a cell proliferation assay on hGM-CSF dependent human erythroleukemia TF-1 cells. This study demonstrated that this indeed is a viable route for the efficient production of hGM-CSF.

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Acknowledgment

The authors are thankful to Prof. R. C. Kuhad, Department of Microbiology, University of Delhi South Campus, for his advice during the preparation of manuscript.

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Authors and Affiliations

  1. Department of Microbiology, University of Delhi South Campus, New Delhi, 110021, India

    Yogender Pal Khasa

  2. School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India

    Yogender Pal Khasa, Amardeep Khushoo, Suman Tapryal & K. J. Mukherjee

  3. Oregon Health and Science University, Portland, 97239, USA

    Amardeep Khushoo

  4. National Institute of Immunology, New Delhi, 110067, India

    Suman Tapryal

Authors
  1. Yogender Pal Khasa
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  2. Amardeep Khushoo
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  3. Suman Tapryal
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  4. K. J. Mukherjee
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Correspondence to K. J. Mukherjee.

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Khasa, Y.P., Khushoo, A., Tapryal, S. et al. Optimization of Human Granulocyte Macrophage-Colony Stimulating Factor (hGM-CSF) Expression Using Asparaginase and Xylanase Gene’s Signal Sequences in Escherichia coli . Appl Biochem Biotechnol 165, 523–537 (2011). https://doi.org/10.1007/s12010-011-9272-5

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  • DOI: https://doi.org/10.1007/s12010-011-9272-5

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