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Vaccinia Virus-Based Expression of gp120 and EGFP:Survey of Mammalian Host Cell Lines

机译:基于痘苗病毒的gp120和EGFP表达:哺乳动物宿主细胞系的调查

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Production of recombinant proteins with the vaccinia virus expression system in five mammalian cell lines (HeLa,BS-C-1,Vero,MRC-5,and 293) was investigated for protein yield and proper posttranslational modifications.Regulatory acceptance of the host cell line was taken into consideration,where Vero,MRC-5,and 293 were considered more acceptable to the regulatory authorities.Relevant process knowledge for ease of scale-up with the particular cell type was also considered.Two proteins were expressed,enhanced green fluorescent protein (EGFP) in the cytoplasm and gp120,an HIV envelope coat protein that is secreted into the culture medium.HeLa cells produced the most EGFP at 17.2mug/well with BS-C-1 and 293 following.BS-C-1 produced the most gp120 at 28.2 mug/mL with 293 and Vero following.Therefore,of the three most appropriate cell lines (Vero,MRC-5,and 293) for production processes,the best results were obtained with 293 cells.Although MRC-5 had a very high productivity on a per cell basis,the low cell density and slow growth rate made the overall production insufficient.Because gp120 contained a significant amount of posttranslational modification,this protein,produced by the different cell lines,was further analyzed by PNGase digestion suggesting N-linked glycosylation modifications in all cell lines tested.On the basis of these results and overall process considerations,293 cells are recommended for further production process optimization in a serum-free suspension system.
机译:研究了牛痘病毒表达系统在5种哺乳动物细胞系(HeLa,BS-C-1,Vero,MRC-5和293)中产生重组蛋白的蛋白质产量和适当的翻译后修饰。考虑到了Vero,MRC-5和293被监管机构接受的程度。还考虑了有关特定细胞类型易于放大的相关工艺知识。表达了两种蛋白质,增强的绿色荧光蛋白质胞质中的(EGFP)和gp120(一种分泌到培养基中的HIV外壳蛋白).HeLa细胞产生的EGFP最高,为17.2mug /孔,随后是BS-C-1和293.BS-C-1产生了gp120的最大浓度为28.2杯/毫升,随后是293和Vero。因此,在三种最适合生产过程的细胞系(Vero,MRC-5和293)中,使用293细胞可获得最佳结果。尽管MRC-5具有每个单元的生产率很高因为gp120含有大量的翻译后修饰,因此该蛋白由不同的细胞系产生,并通过PNGase酶切分析进一步分析,表明所有蛋白中都存在N-联糖基化修饰。根据这些结果和整个过程的考虑,建议将293细胞用于无血清悬浮液系统中的进一步生产工艺优化。

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