Our previous study revealed that N-acetyl-l-cysteine (NAC) could enhance the secretion of recombinant proteins by Pichia pastoris, but the corresponding molecular mechanisms are still unclear. In the present study, we explored whether other thiols have a similar action on the secretion of recombinant human serum albumin and porcine follicle-stimulating hormone fusion protein (HSA-pFSHβ), to reveal the mechanism of NAC on HSA-pFSHβ secretion. Transcriptome analysis showed that genes involved in oxidoreductase activity and oxidation–reduction process were upregulated in cells supplemented with NAC. The other three thiol-reducing regents including dimercaptopropanol (DT), thioglycolic acid, and mercaptolactic acid could improve HSA-pFSHβ production in the culture supernatant. Among them, only DT had similar effect as NAC on HSA-pFSHβ secretion and the increase of GSH content. Moreover, 1–20 mM GSH, 1–10 mM cysteine, or 1–20 mM N-acetyl-d-cysteine supplementation could improve the secretion of HSA-pFSHβ. Furthermore, 0.4–3.2 mM ethacrynic acid, rather than 1–16 mM BSO could inhibit the effect of NAC on the production of HSA-pFSHβ. These results indicated that NAC improved the secretion of HSA-pFSHβ by increasing the intracellular GSH content through its thiol activity rather than as a precursor for GSH synthesis. In conclusion, our results demonstrate, for the first time, that the secretion of recombinant HSA-pFSHβ in Pichia pastoris could be improved through thiol-reducing agent supplementation, and the mechanism of the effect NAC has on HSA-pFSHβ secretion is associated with improving the intracellular GSH content.

中文翻译：

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N-乙酰-l-半胱氨酸促进毕赤酵母分泌猪促卵泡激素的机制

我们之前的研究表明，N-乙酰-l-半胱氨酸（NAC）可以增强毕赤酵母重组蛋白的分泌，但相应的分子机制仍不清楚。在本研究中，我们探讨了其他硫醇是否对重组人血清白蛋白和猪促卵泡激素融合蛋白（HSA-pFSHβ）的分泌具有相似的作用，以揭示NAC对HSA-pFSHβ分泌的作用机制。转录组分析表明，在补充有 NAC 的细胞中，参与氧化还原酶活性和氧化还原过程的基因上调。其他三种硫醇还原剂，包括二巯基丙醇 (DT)、巯基乙酸和巯基乳酸，可以提高培养上清液中 HSA-pFSHβ 的产生。其中，只有DT对HSA-pFSHβ分泌和GSH含量增加的作用与NAC相似。此外，1-20 mM GSH、1-10 mM 半胱氨酸或 1-20 mM N-乙酰-d-半胱氨酸补充可以改善HSA-pFSHβ的分泌。此外，0.4-3.2 mM 依他丙烯酸，而不是 1-16 mM BSO 可以抑制 NAC 对 HSA-pFSHβ 产生的影响。这些结果表明，NAC 通过其硫醇活性而不是作为 GSH 合成的前体来增加细胞内 GSH 含量，从而改善 HSA-pFSHβ 的分泌。总之，我们的研究结果首次表明，通过添加硫醇还原剂可以改善毕赤酵母中重组 HSA-pFSHβ 的分泌， NAC 对 HSA-pFSHβ 分泌的影响机制与改善细胞内 GSH 含量。