N-Glycosylation is Essential for the Expression of Recombinant Human Interleukin-40 in Komagataella phaffii.

Authors

Mayurachat Kaewmanee, Methaphon Paenthong, Juthakorn Phoothong, Phakjira Tiraratchailert, Sawanya Charoenlappanit, Sittiruk Roytrakul, Teva Phanaksri

Published in

Protein expression and purification. Pages 107022. Oct 05, 2026. Epub Oct 05, 2026.

Abstract

Interleukin-40 (IL-40) is a novel cytokine involved in IgA production and B-cell development. Currently, the investigation of recombinant human IL-40 (rhIL-40) is limited to functional studies. In this study, we expressed rhIL-40 and optimized conditions in the methylotrophic yeast, Komagataella phaffii. We introduced the pPICZαA plasmid containing a mature human IL-40 into K. phaffii KM71H by electroporation, screened the transformants on Zeocin™ gradients, and confirmed gene integration by PCR. The secretory expression level of rhIL-40 was approximately 187.30 mg/L after 48 h of induction by 1% (v/v) methanol and addition of phenylmethylsulfonyl fluoride and pepstatin A to the culture medium at 28 °C. Interestingly, rhIL-40 was high molecular weight with N-linked glycans, indicating reduction of the molecular weight to the expected 28 kDa after PNGase F digestion. We also investigated the effect of N-linked glycosylation on rhIL-40 expression and secretion. A single asparagine mutation (N86A or N132A) negatively affected rhIL-40 expression and secretion levels. Moreover, the double mutation revealed a dramatic reduction in rhIL-40 expression and secretion, with little accumulation of the expected intracellular non-glycosylated rhIL-40, suggesting that N-glycosylation mainly influenced the stability of rhIL-40, leading to a lower level of secretory rhIL-40. Our results indicate the feasibility of using K. phaffii KM71H for rhIL-40 expression and the importance of N-linked glycosylation for rhIL-40 stability in the yeast expression system.

PMID:
42833499
Bibliographic data and abstract were imported from PubMed on 06 Oct 2026.

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