Department of Life Science Engineering (LSE), Faculty of New Sciences and Technologies (FNST), University of Tehran, Tehran, Iran.
Department of Quality Control, Research and Production Complex, Pasteur Institute of Iran, Tehran, Iran.
Mol Biotechnol. 2019 Jul;61(7):521-527. doi: 10.1007/s12033-019-00182-6.
The production of the recombinant proteins under the control of AOX1 promoter is a one of the most common expression systems in the methylotrophic yeast Pichia pastoris which is induced by methanol. The application of this expression platform is restricted by the toxicity and inflammatory nature of methanol, especially in food and pharmaceutical products. Human granulocyte macrophage-colony stimulating factor (hGM-CSF) is an important pharmaceutical protein, playing a crucial role in the proliferation and differentiation of innate immune cells. In this study, a methanol-free expression platform for extracellular expression of hGM-CSF was developed. To attain this goal, a novel constructed expression vector pEP(α)101, carrying the FMD promoter regulating recombinant expression by glycerol derepression was designed. The optimized hGM-CSF gene was subcloned into pEP(α)101 and transformed into P. pastoris. The expression of rhGM-CSF in three different culture media were investigated. Based on the observed heterogeneous glycosylation pattern on SDS-PAGE and western blot, the glycoproteins were deglycosylated to remove carbohydrate units. According to the results, the novel methanol independent P expression platform would be a suitable candidate for driving heterologous gene expression especially for the production of food-grade and therapeutically important recombinant proteins.
在甲醇诱导的甲醇营养型酵母毕赤酵母中，受 AOX1 启动子控制的重组蛋白生产是最常见的表达系统之一。该表达平台的应用受到甲醇的毒性和炎症特性的限制，特别是在食品和医药产品中。人粒细胞巨噬细胞集落刺激因子（hGM-CSF）是一种重要的药物蛋白，在先天免疫细胞的增殖和分化中起着关键作用。在本研究中，开发了一种用于 hGM-CSF 细胞外表达的无甲醇表达平台。为了实现这一目标，设计了一种新型构建的表达载体 pEP(α)101，该载体携带 FMD 启动子，通过甘油去阻遏调节重组表达。优化后的 hGM-CSF 基因被亚克隆到 pEP(α)101 中，并转化到毕赤酵母中。在三种不同的培养基中研究了 rhGM-CSF 的表达。根据在 SDS-PAGE 和 western blot 上观察到的不均一糖基化模式，对糖蛋白进行了糖基化去除，以去除碳水化合物单位。根据结果，新型甲醇非依赖性 P 表达平台将是驱动异源基因表达的合适候选者，特别是对于生产食品级和治疗上重要的重组蛋白。
