Ejike Udochukwu Camillius, Chan Chong Joo, Lim Crystale Siew Ying, Lim Renee Lay Hong
Department of Biotechnology, Faculty of Applied Sciences, UCSI University, No.1, Jalan Menara Gading, UCSI Heights, 56000, Cheras, Wilayah Persekutuan Kuala Lumpur, Malaysia.
Appl Microbiol Biotechnol. 2021 Apr;105(7):2799-2813. doi: 10.1007/s00253-021-11225-x. Epub 2021 Mar 25.
Fungal immunomodulatory proteins (FIPs) are bioactive proteins with immunomodulatory properties. We previously reported the heterologous production in Escherichia coli of FIP-Lrh from Tiger milk mushroom (Lignosus rhinocerus) with potent cytotoxic effect on cancer cell lines. However, protein produced in E. coli lacks post-translational modifications and may be contaminated with lipopolysaccharide (LPS) endotoxin. Therefore, in this study, yFIP-Lrh produced in Pichia pastoris was functionally compared with eFIP-Lrh produced in E. coli. Expression construct of FIP-Lrh cDNA in pPICZα was generated, transformed into P. pastoris X-33 and Mut transformants were verified by colony PCR. Induction with 0.5% or 1% methanol resulted in a secreted 13.6 kDa yFIP-Lrh which was subsequently purified and verified using LCMS/MS analysis. Size exclusion chromatography confirmed eFIP-Lrh as a homodimer whereas the larger size of yFIP-Lrh may indicate post-translational modification despite negative for glycoproteins staining. At lower concentration (4-8 μg/mL), yFIP-Lrh induced significantly higher Th1 (IFN-γ, TNF-α) and Th2 (IL-6, IL-4, IL-5, IL-13) cytokines production in mice splenocytes, whereas 16 μg/mL eFIP-Lrh induced significantly higher pro-inflammatory cytokines (TNF-α, IL-6, IL-10), possibly due to higher residual LPS endotoxin (0.082 EU/mL) in eFIP-Lrh compared to negligible level in yFIP-Lrh (0.001 EU/mL). Furthermore, yFIP-Lrh showed higher cytotoxic effect on MCF-7 and HeLa cancer cells. Since both recombinant proteins of FIP-Lrh have the same peptide sequence, besides glycosylation, other post-translational modifications in yFIP-Lrh may account for its enhanced immunomodulatory and anti-proliferative activities. In conclusion, P. pastoris is preferred over E. coli for production of a functionally active yFIP-Lrh devoid of endotoxin contamination. KEY POINTS: • FIP-Lrh can induced production of Th1 and Th2 cytokines by mouse splenocytes. • Higher cytotoxic effect on cancer cells observed for yeast compared to E. coli produced FIP-Lrh. • P. pastoris allows production of an endotoxin-free and functionally active recombinant FIP-Lrh.
真菌免疫调节蛋白（FIPs）是具有免疫调节特性的生物活性蛋白。我们之前报道了从虎奶菇（Lignosus rhinocerus）中提取的FIP-Lrh在大肠杆菌中的异源生产，其对癌细胞系具有强大的细胞毒性作用。然而，在大肠杆菌中产生的蛋白质缺乏翻译后修饰，并且可能被脂多糖（LPS）内毒素污染。因此，在本研究中，对毕赤酵母中产生的yFIP-Lrh与大肠杆菌中产生的eFIP-Lrh进行了功能比较。构建了FIP-Lrh cDNA在pPICZα中的表达载体，将其转化到毕赤酵母X-33中，并通过菌落PCR验证了Mut转化体。用0.5%或1%的甲醇诱导产生了一种分泌型的13.6 kDa yFIP-Lrh，随后对其进行纯化，并使用LCMS/MS分析进行验证。尺寸排阻色谱法证实eFIP-Lrh为同二聚体，而yFIP-Lrh较大的尺寸可能表明存在翻译后修饰，尽管糖蛋白染色呈阴性。在较低浓度（4-8μg/mL）下，yFIP-Lrh在小鼠脾细胞中诱导产生的Th1（IFN-γ、TNF-α）和Th2（IL-6、IL-4、IL-5、IL-13）细胞因子显著更高，而16μg/mL的eFIP-Lrh诱导产生的促炎细胞因子（TNF-α、IL-6、IL-10）显著更高，这可能是由于eFIP-Lrh中残留的LPS内毒素水平较高（0.082 EU/mL），而yFIP-Lrh中的内毒素水平可忽略不计（0.001 EU/mL）。此外，yFIP-Lrh对MCF-7和HeLa癌细胞显示出更高的细胞毒性作用。由于FIP-Lrh的两种重组蛋白具有相同的肽序列，除了糖基化外，yFIP-Lrh中的其他翻译后修饰可能是其增强的免疫调节和抗增殖活性的原因。总之，对于生产无内毒素污染的功能活性yFIP-Lrh，毕赤酵母比大肠杆菌更具优势。要点：• FIP-Lrh可诱导小鼠脾细胞产生Th1和Th2细胞因子。• 与大肠杆菌产生的FIP-Lrh相比，酵母产生的FIP-Lrh对癌细胞的细胞毒性作用更高。• 毕赤酵母可生产无内毒素且功能活性的重组FIP-Lrh。
