Comparison of internal ribosome entry site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells.
Comparison of internal ribosome entry site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells.
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DOI：
10.1371/journal.pone.0063247
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Yang Y
中科院分区：
文献类型：
--
作者：
Ho SC;Bardor M;Li B;Lee JJ;Song Z;Tong YW;Goh LT;Yang Y
设计了4种表达IgG 1轻链（LC）、IgG 1重链（HC）和二氢叶酸还原酶（DHFR）的三顺反子载体，比较了内部核糖体进入位点（IRES）和弗林蛋白酶-2A（F2 A）对单克隆抗体（mAb）在CHO DG 44细胞中表达水平和质量的影响。LC和HC基因排列为第一或第二顺反子。当使用基于针对HC Fc区的检测抗体的mAb定量方法时，在瞬时和稳定转染中，F2 A介导的三顺反子载体似乎以比IRES介导的三顺反子载体更高的水平表达mAb。进一步的分析显示，在使用两种F2 A介导的三顺反子载体产生的稳定转染的池中检测到的超过40%的产物是聚集体。来自F2 A稳定转染池的LC和HC未正确处理，产生LC+ F2 A +HC或HC+ F2 A +LC融合蛋白、具有F2 A残留物的LC和HC多肽以及不正确切割的信号肽。两种IRES介导的三顺反子载体均表达具有正确大小和信号肽切割的mAb。在IRES介导的三顺反子载体中，LC作为第一顺反子的排列表现出增加的mAb表达水平、更好的生长和最小化的产物聚集，而HC作为第一顺反子的排列导致低表达、较慢的生长和高聚集。这些结果将有助于设计提高mAb在哺乳动物细胞中表达水平和质量的载体。
Four versions of tricistronic vectors expressing IgG1 light chain (LC), IgG1 heavy chain (HC), and dihydrofolate reductase (DHFR) in one transcript were designed to compare internal ribosome entry site (IRES) and furin-2A (F2A) for their influence on monoclonal antibody (mAb) expression level and quality in CHO DG44 cells. LC and HC genes are arranged as either the first or the second cistron. When using mAb quantification methods based on the detection antibodies against HC Fc region, F2A-mediated tricistronic vectors appeared to express mAb at higher levels than the IRES-mediated tricistronic vectors in both transient and stable transfections. Further analysis revealed that more than 40% of products detected in stably transfected pools generated using the two F2A-mediated tricistronic vectors were aggregates. LC and HC from the F2A stably transfected pools were not properly processed, giving rise to LC+F2A+HC or HC+F2A+LC fusion proteins, LC and HC polypeptides with F2A remnants, and incorrectly cleaved signal peptides. Both IRES-mediated tricistronic vectors express mAb with correct sizes and signal peptide cleavage. Arrangement of LC as the first cistron in the IRES-mediated tricistronic vectors exhibits increased mAb expression level, better growth, and minimized product aggregation, while arrangement of HC as first cistron results in low expression, slower growth, and high aggregation. The results obtained will be beneficial for designing vectors that enhance mAb expression level and quality in mammalian cells.
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