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Bioprocess development for extracellular production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris

  • Fermentation, Cell Culture and Bioengineering - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Human interleukin-3 (hIL-3) is a therapeutically important cytokine involved in the maturation and differentiation of various cells of the immune system. The codon-optimized hIL-3 gene was cloned in fusion with the N-terminus α-mating factor signal peptide of Saccharomyces cerevisiae under an inducible alcohol oxidase 1 (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. A Zeocin concentration up to 2000 mg/L was used to select hyper-producers. The shake flask cultivation studies in the Pichia pastoris GS115 host resulted a maximum recombinant hIL-3 expression level of 145 mg/L in the extracellular medium under the control of AOX1 promoter. The batch fermentation strategy allowed us to attain a fairly pure glycosylated hIL-3 protein in the culture supernatant at a final concentration of 475 mg/L with a high volumetric productivity of 4.39 mg/L/h. The volumetric product concentration achieved at bioreactor level was 3.28 folds greater than the shake flask results. The 6x His-tagged protein was purified using Ni–NTA affinity chromatography and confirmed further by western blot analysis using anti-6x His tag antibody. The glycosylation of recombinant hIL-3 protein was confirmed in a PNGase F deglycosylation reaction where it showed a molecular weight band pattern similar to E. coli produced non-glycosylated hIL-3 protein. The structural properties of recombinant hIL-3 protein were confirmed by CD and fluorescence spectroscopy where protein showed 40 % α-helix, 12 % β-sheets with an emission maxima at 343 nm. MALDI-TOF-TOF analysis was used to establish the protein identity. The biological activity of purified protein was confirmed by the human erythroleukemia TF-1 cell proliferation assay.

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Acknowledgments

This work is supported by Department of Biotechnology (DBT), Government of India, New Delhi (Grant No. BT/PR5822/PID/6/684/2012). Vikas Kumar Dagar, Adivitiya, and Nirmala Devi are the recipients of a senior research fellowship from Indian Council of Medical Research (ICMR), Council of Scientific and Industrial Research (CSIR) and University Grants Commission (UGC) (Govt. of India) respectively.

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  1. Department of Microbiology, University of Delhi South Campus, New Delhi, 110 021, India

    Vikas Kumar Dagar,  Adivitiya, Nirmala Devi & Yogender Pal Khasa

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  1. Vikas Kumar Dagar
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  2. Adivitiya
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  3. Nirmala Devi
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  4. Yogender Pal Khasa
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Correspondence to Yogender Pal Khasa.

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Dagar, V.K., Adivitiya, Devi, N. et al. Bioprocess development for extracellular production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris . J Ind Microbiol Biotechnol 43, 1373–1386 (2016). https://doi.org/10.1007/s10295-016-1816-9

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  • DOI: https://doi.org/10.1007/s10295-016-1816-9

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