Department of Chemical Engineering, University of California, Davis, CA 95616, USA.
Department of Chemistry, University of California, Davis, CA 95616, USA.
Int J Mol Sci. 2020 Sep 20;21(18):6896. doi: 10.3390/ijms21186896.
The production and -glycosylation of recombinant human butyrylcholinesterase (BChE), a model highly glycosylated therapeutic protein, in a transgenic rice cell suspension culture treated with kifunensine, a strong α-mannosidase I inhibitor, was studied in a 5 L bioreactor. A media exchange was performed at day 7 of cultivation by removing spent sugar-rich medium (NB+S) and adding fresh sugar-free (NB-S) medium to induce the rice α-amylase 3D (RAmy3D) promoter to produce rice recombinant human BChE (rrBChE). Using a 1.25X-concentrated sugar-free medium together with an 80% reduced working volume during the media exchange led to a total active rrBChE production level of 79 ± 2 µg (g FW) or 7.5 ± 0.4 mg L in the presence of kifunensine, which was 1.5-times higher than our previous bioreactor runs using normal sugar-free (NB-S) media with no kifunensine treatment. Importantly, the amount of secreted active rrBChE in culture medium was enhanced in the presence of kifunensine, comprising 44% of the total active rrBChE at day 5 following induction. Coomassie-stained SDS-PAGE gel and Western blot analyses revealed different electrophoretic migration of purified rrBChE bands with and without kifunensine treatment, which was attributed to different -glycoforms. -Glycosylation analysis showed substantially increased oligomannose glycans (Man5/6/7/8) in rrBChE treated with kifunensine compared to controls. However, the mass-transfer limitation of kifunensine was likely the major reason for incomplete inhibition of α-mannosidase I in this bioreactor study.
在 5L 生物反应器中，研究了用 kifunensine（一种强 α-甘露糖苷酶 I 抑制剂）处理的转基因水稻细胞悬浮培养物中重组人丁酰胆碱酯酶（BChE）的产生和 -糖基化，BChE 是一种高度糖基化的治疗蛋白模型。在培养的第 7 天进行了培养基交换，通过去除富含糖的旧培养基（NB+S）并添加新鲜无糖（NB-S）培养基来诱导水稻 α-淀粉酶 3D（RAmy3D）启动子产生水稻重组人 BChE（rrBChE）。使用浓缩 1.25 倍的无糖培养基并在培养基交换期间将工作体积减少 80%，导致在有 kifunensine 的情况下，总活性 rrBChE 的生产水平达到 79±2μg（gFW）或 7.5±0.4mg L，比我们之前使用正常无糖（NB-S）培养基和无 kifunensine 处理的生物反应器运行高出 1.5 倍。重要的是，在有 kifunensine 的情况下，培养基中分泌的活性 rrBChE 量增加，诱导后第 5 天占总活性 rrBChE 的 44%。考马斯亮蓝染色 SDS-PAGE 凝胶和 Western blot 分析显示，有和没有 kifunensine 处理的纯化 rrBChE 带的电泳迁移不同，这归因于不同的 -糖型。-糖基化分析表明，用 kifunensine 处理的 rrBChE 中的寡甘露糖聚糖（Man5/6/7/8）明显增加，与对照相比。然而，kifunensine 的质量传递限制可能是该生物反应器研究中不完全抑制α-甘露糖苷酶 I 的主要原因。
