Engineering of Chinese Hamster Ovary Cells With NDPK-A to Enhance DNA Nuclear Delivery Combined With EBNA1 Plasmid Maintenance Gives Improved Exogenous Transient Reporter, mAb and SARS-CoV-2 Spike Protein Expression.
Engineering of Chinese Hamster Ovary Cells With NDPK-A to Enhance DNA Nuclear Delivery Combined With EBNA1 Plasmid Maintenance Gives Improved Exogenous Transient Reporter, mAb and SARS-CoV-2 Spike Protein Expression.
复制标题
DOI：
10.3389/fbioe.2021.679448
复制
发表时间：
2021
影响因子：
5.7
通讯作者：
Smales CM
中科院分区：
文献类型：
--
作者：
Budge JD;Young RJ;Smales CM
哺乳动物细胞中的瞬时基因表达 (TGE) 是一种快速生成重组蛋白材料的方法，用于初始表征研究，不需要与稳定细胞系构建相关的耗时过程。高 TGE 产量在很大程度上取决于质粒 DNA 跨质膜和核膜的有效传递。在这里，我们利用含有核定位信号 (NLS) 的蛋白质核苷二磷酸激酶 (NDPK-A) 来增强 DNA 递送到 CHO 细胞核中。我们表明，瞬时表达过程中 NDPK-A 的共表达可提高 CHO 细胞的转染效率，这可能是由于增强了质粒 DNA 通过核孔复合物向细胞核的转运。此外，引入 Epstein Barr 核抗原-1 (EBNA-1)（一种能够诱导染色体外维持的蛋白质），当与瞬时质粒上的互补 oriP 元件偶联时，可用于减少质粒稀释的影响。虽然将 EBNA-1 系统引入 CHO 细胞后生长减弱，但当同时使用 NDPK-A 核导入和 EBNA-1 介导技术时，这会导致瞬时重组蛋白产量增强，优于单独使用任一方法产生的产量，包括在表达复杂的 SARS-CoV-2 刺突 (S) 糖蛋白时。
Transient gene expression (TGE) in mammalian cells is a method of rapidly generating recombinant protein material for initial characterisation studies that does not require time-consuming processes associated with stable cell line construction. High TGE yields are heavily dependent on efficient delivery of plasmid DNA across both the plasma and nuclear membranes. Here, we harness the protein nucleoside diphosphate kinase (NDPK-A) that contains a nuclear localisation signal (NLS) to enhance DNA delivery into the nucleus of CHO cells. We show that co-expression of NDPK-A during transient expression results in improved transfection efficiency in CHO cells, presumably due to enhanced transportation of plasmid DNA into the nucleus via the nuclear pore complex. Furthermore, introduction of the Epstein Barr Nuclear Antigen-1 (EBNA-1), a protein that is capable of inducing extrachromosomal maintenance, when coupled with complementary oriP elements on a transient plasmid, was utilised to reduce the effect of plasmid dilution. Whilst there was attenuated growth upon introduction of the EBNA-1 system into CHO cells, when both NDPK-A nuclear import and EBNA-1 mediated technologies were employed together this resulted in enhanced transient recombinant protein yields superior to those generated using either approach independently, including when expressing the complex SARS-CoV-2 spike (S) glycoprotein.
登录
查看更多内容
影响因子：
3.8
作者：
Kunaparaju, R;Liao, M;Sunstrom, NA
通讯作者：
Sunstrom, NA
影响因子：
14.9
作者：
Kreiss, P;Cameron, B;Pitard, B
通讯作者：
Pitard, B
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：
3.7
作者：
Dean, DA
通讯作者：
Dean, DA
DOI：
10.1126/science.1253768
发表时间：
2014-06-27
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Boissan M;Montagnac G;Shen Q;Griparic L;Guitton J;Romao M;Sauvonnet N;Lagache T;Lascu I;Raposo G;Desbourdes C;Schlattner U;Lacombe ML;Polo S;van der Bliek AM;Roux A;Chavrier P
通讯作者：
Chavrier P