Simple piggyBac transposon-based mammalian cell expression system for inducible protein production
Simple piggyBac transposon-based mammalian cell expression system for inducible protein production
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DOI：
10.1073/pnas.1218620110
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发表时间：
2013-03-26
影响因子：
11.1
通讯作者：
Rini, James M.
中科院分区：
文献类型：
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作者：
Li, Zhijie;Michael, Iacovos P.;Rini, James M.
本文报道了一种基于piggyBac转座子的表达系统，用于产生多西环素诱导的、稳定转染的哺乳动物细胞培养物，用于大规模蛋白质生产。该系统与常用的粘附和悬浮适应的哺乳动物细胞系一起工作，并且仅需要单个转染步骤。此外，在克隆中观察到的高均匀表达水平允许使用稳定的大量细胞培养物，从而消除耗时的克隆步骤。在连续强力霉素诱导下，蛋白质表达水平已显示在没有药物选择的情况下稳定至少2个月。该系统的高效率还允许以96孔格式产生稳定的大量细胞培养物，这一能力导致产生膜或分泌蛋白的整个家族的稳定细胞培养物的可能性。最后，我们通过大规模生产（140-750毫克规模）的内质网驻留岩藻糖基转移酶和两个潜在的抗癌蛋白质治疗剂证明了该系统的实用性。
Reported here is a piggyBac transposon-based expression system for the generation of doxycycline-inducible, stably transfected mammalian cell cultures for large-scale protein production. The system works with commonly used adherent and suspension-adapted mammalian cell lines and requires only a single transfection step. Moreover, the high uniform expression levels observed among clones allow for the use of stable bulk cell cultures, thereby eliminating time-consuming cloning steps. Under continuous doxycycline induction, protein expression levels have been shown to be stable for at least 2 mo in the absence of drug selection. The high efficiency of the system also allows for the generation of stable bulk cell cultures in 96-well format, a capability leading to the possibility of generating stable cell cultures for entire families of membrane or secreted proteins. Finally, we demonstrate the utility of the system through the large-scale production (140-750 mg scale) of an endoplasmic reticulum-resident fucosyltransferase and two potential anticancer protein therapeutic agents.