Enhancement of recombinant human interleukin-22 production by fusing with human serum albumin and supplementing N-acetylcysteine in Pichia Pastoris.

Interleukin-22 (IL-22) plays an important role in the treatment of organ failure, which can induce anti-apoptotic and proliferative signaling pathways; Nevertheless, the practical utilization of IL-22 is hindered by the restricted efficacy of its production. Pichia pastoris presents a viable platform for both industrial and pharmaceutical applications. In this study, we successfully generated a fusion protein consisting of truncated human serum albumin and human IL-22 (HSA-hIL-22) using P. pastoris, and examined the impact of antioxidants on HSA-hIL-22 production. We have achieved the production of HSA-hIL-22 in the culture medium at a yield of approximately 2.25 mg/ml. Moreover, 0-40 mM ascorbic acid supplementation did not significantly affect HSA-hIL-22 production or the growth rate of the recombinant strain. However, 80 mM ascorbic acid treatment had a detrimental effect on the expression of HSA-hIL-22. In addition, 5-10 mM N-acetyl-l-cysteine (NAC) resulted in an increase of HSA-hIL-22 production, accompanied by a reduction in the growth rate of the recombinant strain. Conversely, 20-80 mM NAC supplementation inhibited the growth of the recombinant strains and reduced intact HSA-hIL-22 production. However, neither NAC nor ascorbic acid exhibited any effect on superoxide dismutase (SOD) and malondialdehyde (MDA) levels, except that NAC increased GSH content. Furthermore, our findings indicate that recombinant HSA-hIL-22, which demonstrated the ability to stimulate the proliferation of HepG2 cells, possesses bioactivity. In addition, NAC did not affect HSA-hIL-22 bioactivity. In conclusion, our study demonstrates that NAC supplementation can enhance the secretion of functional HSA-hIL-22 proteins produced in P. pastoris without compromising their activity.

Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein.

Pichia pastoris is widely used for the production of recombinant proteins, but the low secretion efficiency hinders its wide application in biopharmaceuticals. Our previous study had shown that N-acetyl-l-cysteine (NAC) promotes human serum albumin and porcine follicle-stimulating hormone fusion protein (HSA-pFSHß) secretion by increasing intracellular GSH levels, but the downstream impact mechanism is not clear. In this study, we investigated the roles of autophagy as well as cell phenotype in NAC promoting HSA-pFSHß secretion. Our results showed that NAC slowed down the cell growth rate, and its effects were unaffected by Congo Red and Calcofluor White. Moreover, NAC affected cell wall composition by increasing chitin content and decreasing ß-1,3-glucan content. In addition, the expressions of vesicular pathway and autophagy-related genes were significantly decreased after NAC treatment. Further studies revealed that autophagy, especially the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy and pexophagy, was significantly increased with time, and NAC has a promoting effect on autophagy, especially at 48 h and 72 h of NAC treatment. However, the disruption of mitophagy receptor Atg32, but not pexophagy receptor Atg30, inhibited HSA-pFSHß production, and neither of them inhibited the NAC-promoted effect of HSA-pFSHß. In conclusion, vesicular transport, autophagy and cell wall are all involved in the NAC-promoted HSA-pFSHß secretion and that disruption of the autophagy receptor alone does not inhibit the effect of NAC.

Enhancement of HSA-pFSHß production by disrupting YPS1 and supplementing N-acetyl-L-cysteine in Pichia pastoris.

Introduction: Pichia pastoris is widely used for the production of recombinant proteins, but the low production efficiency hinders its wide application in biopharmaceuticals. Moreover, many biopharmaceutical-like proteins are accompanied by degradation during secretory expression in P. pastoris. Objective: In this study, we used human serum albumin and porcine follicle-stimulating hormone ß (HSA-pFSHß) fusion protein as a model protein to investigate whether YPS1 and YPT7 gene disruption and N-acetyl-L-cysteine (NAC) supplementation have synergistic effects to inhibit the degradation of recombinant proteins. Results and discussion: Our results showed that YPS1 gene disruption reduced the degradation of intact HSA-pFSHß and increased the yield of intact protein in the culture medium and cells without affecting the integrity of the cell wall. Moreover, the beneficial effects of YPS1 gene disruption were associated with the upregulation of the MAPK signaling pathway and maintenance of redox homeostasis. YPS1 gene disruption and NAC supplementation had synergistic effects on HSA-pFSHß production. In addition, disruption of vacuolar morphology by YPT7 gene disruption or NH4Cl treatment affected the production of recombinant HSA-pFSHß protein. Furthermore, YPT7 gene disruption inhibited the processing of signal peptide in high-level produced HSA-pFSHß strain. In conclusion, our results demonstrated that YPS1 disruption could reduce the degradation of intact HSA-pFSHß proteins, and synergistically increase the yield of intact HSA-pFSHß with NAC supplementation. This study provided a valuable reference for reducing recombinant protein degradation and therefore improving the yield of recombinant proteins in P. pastoris.