Attenuation of glutamine synthetase selection marker improves product titer and reduces glutamine overflow in Chinese hamster ovary cells
Attenuation of glutamine synthetase selection marker improves product titer and reduces glutamine overflow in Chinese hamster ovary cells
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谷氨酰胺合成酶选择标记的减弱可提高中国仓鼠卵巢细胞中的产物滴度并减少谷氨酰胺溢出
DOI：
10.1002/bit.28084
复制
发表时间：
2022
影响因子：
3.8
通讯作者：
Smith, Kevin D.
中科院分区：
文献类型：
--
作者：
Sacco, Sarah A.;Tuckowski, Angela M.;Trenary, Irina;Kraft, Lauren;Betenbaugh, Michael J.;Young, Jamey D.;Smith, Kevin D.
谷氨酰胺合成酶(GS)表达系统是确保转基因在中国仓鼠卵巢(CHO)宿主系中稳定整合和扩增的常用方法。转基因细胞群体通常在GS抑制剂蛋氨酸亚磺胺(MSX)存在的情况下培养，以进一步选择增加的转基因拷贝数。然而，高水平的GS活性会产生过量的谷氨酰胺。我们推测，减弱GS启动子的同时保持GS-Ig G表达载体上的强Ig G启动子将导致更有效的细胞代谢表型。在这里，我们对表达GS的CHO细胞系进行了鉴定，这些细胞系来自弱化启动子或SV40启动子，并选择了有或没有MSX的细胞株。与SV40驱动的GS表达细胞相比，具有减弱的GS启动子的CHO细胞具有更高的免疫球蛋白特异性生产力和更低的谷氨酰胺产量。无论GS启动子的强度如何，MSX的选择都可以提高比生产力和谷氨酰胺产量。13C代谢通量分析(MFA)进一步评估了这些细胞系之间的代谢差异。有趣的是，减弱的GS启动子没有改变中心碳代谢，而谷氨酸和谷氨酰胺的命运取决于启动子的强度和选择条件。这项研究强调了优化GS表达系统以提高IgG产量和减少谷氨酰胺浪费溢出的能力，而不会显著改变中枢代谢。此外，对两个“乳酸失控”反应器的详细补充分析提供了对一些CHO细胞培养物产生过量乳酸这一鲜为人知的现象的洞察。
The glutamine synthetase (GS) expression system is commonly used to ensure stable transgene integration and amplification in Chinese hamster ovary (CHO) host lines. Transfected cell populations are typically grown in the presence of the GS inhibitor, methionine sulfoximine (MSX), to further select for increased transgene copy number. However, high levels of GS activity produce excess glutamine. We hypothesized that attenuating the GS promoter while keeping the strong IgG promoter on the GS‐IgG expression vector would result in a more efficient cellular metabolic phenotype. Herein, we characterized CHO cell lines expressing GS from either an attenuated promoter or an SV40 promoter and selected with/without MSX. CHO cells with the attenuated GS promoter had higher IgG specific productivity and lower glutamine production compared to cells with SV40‐driven GS expression. Selection with MSX increased both specific productivity and glutamine production, regardless of GS promoter strength.13C metabolic flux analysis (MFA) was performed to further assess metabolic differences between these cell lines. Interestingly, central carbon metabolism was unaltered by the attenuated GS promoter while the fate of glutamate and glutamine varied depending on promoter strength and selection conditions. This study highlights the ability to optimize the GS expression system to improve IgG production and reduce wasteful glutamine overflow, without significantly altering central metabolism. Additionally, a detailed supplementary analysis of two “lactate runaway” reactors provides insight into the poorly understood phenomenon of excess lactate production by some CHO cell cultures.
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DOI：
--
发表时间：
2017
期刊：
影响因子：
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作者：
Wichaya Pripatnanont;J. Opanuraks;K. Prasopsanti;A. Santi;Supoj Ratchanon;K. Tantiwongse;K. Panumatrassamee;Dutsadee Sowanthip
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3.8
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Fan, Lianchun;Kadura, Ibrahim;Frye, Christopher C.
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DOI：
--
发表时间：
2020
期刊：
影响因子：
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作者：
Masato Takahashi;Hideki Aoyagi;Takeo Taniguchi Nobuyuki Okahashi Fumio Matsuda
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Takeo Taniguchi Nobuyuki Okahashi Fumio Matsuda