Inhibition of 2A-mediated 'cleavage' of certain artificial polyproteins bearing N-terminal signal sequences.
Inhibition of 2A-mediated 'cleavage' of certain artificial polyproteins bearing N-terminal signal sequences.
复制标题
DOI：
10.1002/biot.200900134
复制
发表时间：
2010-02
影响因子：
4.7
通讯作者：
Ryan, Martin D.
中科院分区：
文献类型：
--
作者：
de Felipe, Pablo;Luke, Garry A.;Brown, Jeremy D.;Ryan, Martin D.
如果2A寡肽序列出现在orf内，则不会在（每个）2A的c端形成甘氨酸-脯氨酸肽键。该特性可用于将编码多个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作为离散的翻译产物生成。2A和“2A样”序列已广泛应用于生物技术和生物医学。单个蛋白质也可以共同和翻译后靶向各种亚细胞位点。然而，在携带n端信号序列的多蛋白中，我们观察到2A下游的蛋白（无信号）被转运到内质网（ER）中。我们将这些数据解释为“滑流”易位的一种形式：下游蛋白质在没有信号的情况下，通过多蛋白n端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转运到内质网时，c端区域（2A的上游）抑制了2A反应（融合蛋白的形成）。解决这个问题的方法包括使用更长的2As（具有有利的上游环境）或修改包含多蛋白的蛋白质的顺序。
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.
登录
查看更多内容
影响因子：
20.3
作者：
Huang, X;Wilber, AC;Zhou, XZ
通讯作者：
Zhou, XZ
影响因子：
4.5
作者：
de Felipe, P;Ryan, MD
通讯作者：
Ryan, MD
影响因子：
46.9
作者：
Fang, JM;Qian, JJ;Jooss, K
通讯作者：
Jooss, K
影响因子：
5.1
作者：
de Felipe, P;Martín, V;Izquierdo, M
通讯作者：
Izquierdo, M
影响因子：
30.5
作者：
Holst, Jeff;Wang, Haopeng;Vignali, Dario A. A.
通讯作者：
Vignali, Dario A. A.