Targeting of proteins derived from self-processing polyproteins containing multiple signal sequences
Targeting of proteins derived from self-processing polyproteins containing multiple signal sequences
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DOI：
10.1111/j.1398-9219.2004.00205.x
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发表时间：
2004-08-01
期刊：
影响因子：
4.5
通讯作者：
Ryan, MD
中科院分区：
文献类型：
--
作者：
de Felipe, P;Ryan, MD
来自口蹄疫病毒的 18aa 2A 自切割寡肽可用于来自单个 ORF 的多个离散蛋白质的共表达。 2A 在其自身的 C 末端介导共翻译切割，并被提议操纵核糖体跳过特定肽键的合成（在肽主链中产生不连续性），而不是参与蛋白水解。为了探索该系统针对离散加工产品的效用，构建了包含侧翼2A的荧光蛋白的自加工多蛋白，排列了信号序列的类型和多蛋白内的位置。构建了包含带有N端信号序列2A的蛋白质、然后缺乏任何信号序列的蛋白质的多蛋白。有趣的是，这两种蛋白质都被转移到内质网中。尽管肽主链不连续，哺乳动物核糖体：易位子复合物并未分解——第二个蛋白质（缺乏任何信号）“滑流”通过第一个（带有信号）蛋白质形成的易位子。这些多蛋白系统提供了一种通过用编码单个 ORF 的质粒转染将蛋白质靶向不同亚细胞位点的新方法。荧光报告基因的加入使得表达水平可视化，而选择性标记的加入使得稳定的细胞系能够快速建立。
The 18aa 2A self-cleaving oligopeptide from foot-and-mouth disease virus can be used for co-expression of multiple, discrete proteins from a single ORF. 2A mediates a co-translational cleavage at its own C-terminus and is proposed to manipulate the ribosome into skipping the synthesis of a specific peptide bond (producing a discontinuity in the peptide backbone), rather than being involved in proteolysis. To explore the utility of the system to target discrete processing products, self-processing polyproteins comprising fluorescent proteins flanking 2A were constructed, permutating both the type of signal sequence and the location within the polyprotein. A polyprotein comprising a protein bearing an N-terminal signal sequence, 2A, then a protein lacking any signal sequence, was constructed. Interestingly, both proteins were translocated into the endoplasmic reticulum. Despite the discontinuity in the peptide backbone, the mammalian ribosome:translocon complex did not disassemble - the second protein (lacking any signal) 'slipstreamed' through the translocon formed by the first (signal-bearing) protein. These polyprotein systems provide a novel method of targeting proteins to different subcellular sites by transfection with a plasmid encoding a single ORF. The inclusion of a fluorescent reporter enables visualisation of expression levels, whilst inclusion of a selectable marker enables stable cell-lines to be established rapidly.