Development of a one-plasmid system to replace the endogenous protein with point mutation for post-translational modification studies
Development of a one-plasmid system to replace the endogenous protein with point mutation for post-translational modification studies
复制标题
开发一种单质粒系统，用点突变替代内源蛋白，用于翻译后修饰研究
DOI：
10.1007/s11033-021-06693-3
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发表时间：
2021-11-11
影响因子：
2.8
通讯作者：
Yang, Zhenye
中科院分区：
文献类型：
--
作者：
Ishrat, Iqra;Cheng, Aoxing;Yang, Zhenye
背景翻译后修饰（Post-translational modification，PTM）是蛋白质活性的主要调控机制之一。为了理解PTM的功能，经常利用防止或模拟修饰的突变体。内源性蛋白质通常被耗尽，而点突变被表达。完成这些任务的常见策略包括两步：首先，产生稳定表达用于蛋白质耗尽的shRNA的细胞系，然后引入RNAi抗性构建体以表达突变体。然而，这些步骤是费时费力的。更重要的是，shRNA和突变蛋白经常在不同的细胞在不同的时间，这显着扰乱的conclusions.MethodsTo克服这些技术问题，我们开发了一个慢病毒为基础的一个质粒系统，允许并发表达的shRNA和突变蛋白。插入嘌呤霉素抗性基因的选择稳定表达cells.ResultsUsing this plasmid，我们有效地取代了内源性蛋白质与外源性蛋白质的LDHB和PKM 2，两个糖酵解酶调节PTM在癌细胞中的可比水平。该系统也被成功地利用在多个在体外和体内assays.ConclusionThus，我们已经开发出一个有效的单质粒系统，以取代内源性蛋白质的点突变PTM的功能研究中的作用，LDHB丝氨酸162磷酸化的评估。
BackgroundPost-translational modification (PTM) is one of the major regulatory mechanism for protein activities. To understand the function of PTMs, mutants that prevent or mimic the modification are frequently utilized. The endogenous proteins are usually depleted while the point mutations are expressed. A common strategy to accomplish these tasks includes two-steps: First, a cell line stably expressing shRNA for protein depletion is generated, then an RNAi-resistance construct is introduced to express mutant. However, these steps are time- and labor-consuming. More importantly, shRNA and mutant protein are frequently expressed in different cells at different time, which significantly disturbs the conclusions.MethodsTo overcome these technical problems, we developed a lentiviral based one-plasmid system that allowed concurrent expression of shRNA and mutant protein. The puromycin-resistant gene was inserted for the selection of stable-expression cells.ResultsUsing this plasmid, we efficiently replaced the endogenous proteins with comparable levels of exogenous proteins for LDHB and PKM2, two glycolytic enzymes regulated by PTM in cancer cells. The system was also successfully exploited in evaluating the role of phosphorylation of LDHB serine 162 in multiple in vitro and in vivo assays.ConclusionThus, we have developed an efficient one-plasmid system to replace endogenous protein with point mutations for the functional study of PTM.