N-glycosylation is essential for the expression of recombinant human interleukin-40 in Komagataella phaffii

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.