Where 2A oligopeptide sequences occur within ORFs, the formation of the glycyl-prolyl peptide bond at the C-terminus of (each) 2A does not occur. This property can be used to concatenate sequences encoding several proteins into a single ORF: each component of such an artificial polyprotein is generated as a discrete translation product. 2A and '2A-like' sequences have become widely utilised in biotechnology and biomedicine. Individual proteins may also be co- and post-translationally targeted to a variety of sub-cellular sites. In the case of polyproteins bearing N-terminal signal sequences we observed, however, that the protein downstream of 2A (no signal) was translocated into the endoplasmic reticulum (ER). We interpreted these data as a form of 'slipstream' translocation: downstream proteins, without signals, were translocated through a translocon pore already formed by the signal sequence at the N-terminus of the polyprotein. Here we show this effect is, in fact, due to inhibition of the 2A reaction (formation of fusion protein) by the C-terminal region (immediately upstream of 2A) of some proteins when translocated into the ER. Solutions to this problem include the use of longer 2As (with a favourable upstream context) or modifying the order of proteins comprising polyproteins.

中文翻译：

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抑制 2A 介导的某些带有 N 端信号序列的人工多蛋白的“切割”。

当 2A 寡肽序列出现在 ORF 内时，不会在（每个）2A 的 C 端形成甘氨酰-脯氨酰肽键。此特性可用于将编码多种蛋白质的序列连接到单个 ORF 中：这种人工多蛋白的每个成分都是作为离散的翻译产物产生的。2A 和“2A 样”序列已广泛用于生物技术和生物医学。单个蛋白质也可以共翻译和翻译后靶向多种亚细胞位点。然而，在带有 N 端信号序列的多蛋白的情况下，我们观察到 2A 下游的蛋白质（无信号）被转移到内质网（ER）中。我们将这些数据解释为一种“滑流”易位形式：下游蛋白质，没有信号，通过多蛋白 N 末端的信号序列已经形成的易位孔进行易位。在这里，我们表明这种效应实际上是由于某些蛋白质在易位到 ER 时的 C 端区域（紧邻 2A 的上游）抑制了 2A 反应（融合蛋白的形成）。这个问题的解决方案包括使用更长的 2A（具有有利的上游环境）或修改包含多蛋白的蛋白质的顺序。