Tran Anh-Minh, Nguyen Thanh-Thao, Nguyen Cong-Thuan, Huynh-Thi Xuan-Mai, Nguyen Cao-Tri, Trinh Minh-Thuong, Tran Linh-Thuoc, Cartwright Stephanie P, Bill Roslyn M, Tran-Van Hieu
Faculty of Biology and Biotechnology, University of Science, VNU-HCM, Ho Chi Minh, Vietnam.
School of Life and Health Sciences, Aston University, Birmingham, UK.
BMC Res Notes. 2017 Apr 4;10(1):148. doi: 10.1186/s13104-017-2471-6.
Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts.
Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1.
Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production.
重组人粒细胞巨噬细胞集落刺激因子（rhGM-CSF）是一种糖蛋白，已获美国食品药品监督管理局（FDA）批准，可与化疗联合用于治疗中性粒细胞减少症和白血病。重组hGM-CSF是通过面包酵母酿酒酵母进行大规模发酵工业生产的。甲醇营养型酵母毕赤酵母已成为一种替代宿主细胞系统，因为它具有更短且免疫原性更低的糖基化模式，同时与酿酒酵母相比具有更高的细胞密度生长和更高的分泌蛋白产量。在本研究中，我们比较了这两种酵母中从基因到重组蛋白的流程。
对两种酵母进行的计算机密码子优化显示常用密码子使用情况无差异。然而，酿酒酵母BY4742表达的rhGM-CSF在分子量上与毕赤酵母X33表达的rhGM-CSF存在显著差异。分析显示纯化的rhGM-CSF分子量范围为30至60 kDa以上。72小时的补料分批发酵表明，毕赤酵母分泌rhGM-CSF的量高于酿酒酵母（分别为总分泌蛋白285和64 mg/L）。离子交换色谱法比疏水相互作用色谱法具有更高的纯度和回收率。就对hGM-CSF依赖细胞系TF-1的增殖刺激能力而言，毕赤酵母纯化的rhGM-CSF比酿酒酵母纯化的rhGM-CSF强327倍。
我们的数据支持这样一种观点，即甲醇营养型酵母毕赤酵母是生产异源rhGM-CSF的有效重组宿主。
