Abstract
Human interleukin-3 (hIL-3) is a clinically important cytokine used to treat hematological malignancies, bone marrow transplantation, cytopenias, and immunological disorders. The cloning of hIL-3 gene was previously reported by our group, where its expression was optimized under methanol-inducible AOX1 promoter having N-terminal α mating factor signal sequence from Saccharomyces cerevisiae. This study investigated the role of glycosylation pattern on its molecular stability, secretion efficiency, and biological activity using the mutagenesis approach. The two N-linked glycosylation positions at N15th (Asn15) and N70th (Asn70) were sequentially mutated to generate three recombinant hIL-3 variants, i.e., N15A, N70A, and N15/70A. Asparagine at these positions was replaced with non-polar alanine amino acid (Ala, A). The alteration of N-linked glycosylation sites was disadvantageous to its efficient secretion in Pichia pastoris, where a 52.32%, 36.48%, 71.41% lower production was observed in N15A, N70A, and N15/70A mutants, respectively, as compared to native control. The fully glycosylated native hIL-3 protein showed higher thermal stability over its deglycosylated counterparts. The biological activity of native, N15A, N70A, and N15/70A hIL-3 protein was evaluated, where N15/70A mutant showed slightly higher proliferation efficacy than other combinations.
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Funding
The research work was supported by Department of Biotechnology (DBT), Government of India, New Delhi via Grant No. BT/PR5822/PID/6/684/2012 to Dr. Yogender Pal Khasa (Project Investigator). Vikas Kumar Dagar, acknowledges the financial support from the Indian Council of Medical Research (ICMR, Govt. of India) New Delhi as he received a senior research fellowship (Sanction No. 3/1/3-JRF-2009/MPD-89).
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Dagar, V.K., Babbal, Mohanty, S. et al. Effect of N-glycosylation on secretion, stability, and biological activity of recombinant human interleukin-3 (hIL-3) in Pichia pastoris. 3 Biotech 12, 221 (2022). https://doi.org/10.1007/s13205-022-03293-1
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DOI: https://doi.org/10.1007/s13205-022-03293-1