Effect of N-glycosylation on secretion, stability, and biological activity of recombinant human interleukin-3 (hIL-3) in Pichia pastoris
© King Abdulaziz City for Science and Technology 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2022 |
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| Erschienen: |
2022 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:12 |
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| Enthalten in: |
3 Biotech - 12(2022), 9 vom: 07. Sept., Seite 221 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Dagar, Vikas Kumar [VerfasserIn] |
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| Links: |
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| Themen: |
Biological activity |
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| Anmerkungen: |
Date Revised 02.09.2023 published: Print-Electronic Citation Status PubMed-not-MEDLINE |
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| doi: |
10.1007/s13205-022-03293-1 |
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| Förderinstitution / Projekttitel: |
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| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Biological activity | |
| 650 | 4 | |a Human interleukin-3 (hIL-3) | |
| 650 | 4 | |a N-glycosylation | |
| 650 | 4 | |a Pichia pastoris | |
| 650 | 4 | |a Post-translational modifications (PTMs) | |
| 650 | 4 | |a Site-directed mutagenesis | |
| 650 | 4 | |a Thermostability | |
| 700 | 1 | |a Babbal |e verfasserin |4 aut | |
| 700 | 1 | |a Mohanty, Shilpa |e verfasserin |4 aut | |
| 700 | 1 | |a Khasa, Yogender Pal |e verfasserin |4 aut | |
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