A proline metabolism selection system and its application to the engineering of lipid biosynthesis in Chinese hamster ovary cells.
A proline metabolism selection system and its application to the engineering of lipid biosynthesis in Chinese hamster ovary cells.
复制标题
DOI：
10.1016/j.mec.2021.e00179
复制
发表时间：
2021-12
影响因子：
5.2
通讯作者：
Smales CM
中科院分区：
文献类型：
--
作者：
Budge JD;Roobol J;Singh G;Mozzanino T;Knight TJ;Povey J;Dean A;Turner SJ;Jaques CM;Young RJ;Racher AJ;Smales CM
中国仓鼠卵巢（CHO）细胞是用于产生复杂分泌的重组生物治疗剂（例如单克隆抗体（mAb））的主要哺乳动物细胞宿主。代谢选择标记技术（例如谷氨酰胺合成酶（GS）或二氢叶酸还原酶（DHFR））通常用于产生这样的重组哺乳动物细胞系。在这里，我们描述了一个选择标记系统的基础上，CHO细胞产生脯氨酸的代谢要求的发展，并使用吡咯啉-5-羧化酶合成酶（P5 CS），以补充这种营养缺陷型。首先，我们表明该系统可用于产生在无脯氨酸培养基中具有与在含脯氨酸培养基中生长的亲本CHO细胞系CHOK 1 SV GS-KO™相似的生长动力学的细胞。如我们先前所描述的，如何利用工程化脂质代谢来增强CHO细胞中的重组蛋白生产率，然后我们使用P5 CS选择系统通过过表达固醇调节元件结合蛋白1（SREBF 1）或硬脂酰CoA去饱和酶1（SCD 1）来重新工程化脂质代谢。具有重新工程化的脯氨酸和脂质代谢的细胞在100代细胞中显示出一致的生长和P5 CS、SCD 1和SREBF 1表达。最后，我们表明，P5 CS和GS选择系统可以一起使用。将含有mAb的轻链和重链的GS载体从P5 CS载体超转染到过表达SCD 1的CHOK 1 SV GS-KO™宿主中。对于难以表达mAb的模型，所得稳定转染子库在收获时达到比CHOK 1 SV GS-KO™宿主更高的浓度。这表明P5 CS和GS选择系统可以同时使用以实现CHO细胞系遗传工程和重组蛋白表达。我们已经在CHO细胞中设计了脯氨酸P5 CS代谢选择系统P5 CS脯氨酸选择用于在CHO细胞中设计脂质代谢P5 CS选择稳定至少100代P5 CS和GS选择系统一起用于设计脂质和mAb表达脂质代谢P5 CS工程化CHO细胞产生增强的重组蛋白表达
Chinese hamster ovary (CHO) cells are the leading mammalian cell host employed to produce complex secreted recombinant biotherapeutics such as monoclonal antibodies (mAbs). Metabolic selection marker technologies (e.g. glutamine synthetase (GS) or dihydrofolate reductase (DHFR)) are routinely employed to generate such recombinant mammalian cell lines. Here we describe the development of a selection marker system based on the metabolic requirement of CHO cells to produce proline, and that uses pyrroline-5-carboxylase synthetase (P5CS) to complement this auxotrophy. Firstly, we showed the system can be used to generate cells that have growth kinetics in proline-free medium similar to those of the parent CHO cell line, CHOK1SV GS-KO™ grown in proline-containing medium. As we have previously described how engineering lipid metabolism can be harnessed to enhance recombinant protein productivity in CHO cells, we then used the P5CS selection system to re-engineer lipid metabolism by over-expression of either sterol regulatory element binding protein 1 (SREBF1) or stearoyl CoA desaturase 1 (SCD1). The cells with re-engineered proline and lipid metabolism showed consistent growth and P5CS, SCD1 and SREBF1 expression across 100 cell generations. Finally, we show that the P5CS and GS selection systems can be used together. A GS vector containing the light and heavy chains for a mAb was super-transfected into a CHOK1SV GS-KO™ host over-expressing SCD1 from a P5CS vector. The resulting stable transfectant pools achieved a higher concentration at harvest for a model difficult to express mAb than the CHOK1SV GS-KO™ host. This demonstrates that the P5CS and GS selection systems can be used concomitantly to enable CHO cell line genetic engineering and recombinant protein expression. We have engineered a proline P5CS metabolism selection system in CHO cells P5CS proline selection was used to engineer lipid metabolism in CHO cells P5CS selection was stable for at least 100 generations P5CS and GS selection systems were used together to engineer lipid and mAb expression Lipid metabolism P5CS engineered CHO cells give enhanced recombinant protein expression
登录
查看更多内容
影响因子：
3.8
作者：
Fan, Lianchun;Kadura, Ibrahim;Frye, Christopher C.
通讯作者：
Frye, Christopher C.
影响因子：
4.8
作者：
Hu, CA;Lin, WW;Valle, D
通讯作者：
Valle, D
影响因子：
4.1
作者：
Povey, Jane F.;O'Malley, Christopher J.;Smales, C. Mark
通讯作者：
Smales, C. Mark
影响因子：
3.3
作者：
Heinemann, FS;Ozols, J
通讯作者：
Ozols, J
影响因子：
4.8
作者：
Roca, Berta Capella;Lao, Nga;Clynes, Martin
通讯作者：
Clynes, Martin