FOOT-AND-MOUTH-DISEASE VIRUS 2A OLIGOPEPTIDE MEDIATED CLEAVAGE OF AN ARTIFICIAL POLYPROTEIN
FOOT-AND-MOUTH-DISEASE VIRUS 2A OLIGOPEPTIDE MEDIATED CLEAVAGE OF AN ARTIFICIAL POLYPROTEIN
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DOI：
10.1002/j.1460-2075.1994.tb06337.x
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发表时间：
1994-02-15
期刊：
影响因子：
11.4
通讯作者：
DREW, J
中科院分区：
文献类型：
--
作者：
RYAN, MD;DREW, J
我们描述了一种编码多聚蛋白的质粒（pCAT2AGUS）的构建。在该质粒中，口蹄疫病毒（FMDV）多聚蛋白的2A区域的一段19个氨基酸序列被插入到报告基因氯霉素乙酰转移酶（CAT）和β - 葡萄糖醛酸苷酶（GUS）之间，保持了一个单一的长开放阅读框。对由该构建体编程的翻译反应的分析表明，插入的FMDV序列的作用方式与在FMDV多聚蛋白加工中观察到的相似：CAT2AGUS多聚蛋白经历了共翻译的、明显的自蛋白水解切割，产生CAT - 2A和GUS。对一系列构建体的翻译产物的分析表明，在这些构建体中，FMDV 2A插入片段的N末端区域的序列逐渐缺失，切割至少需要13个残基。因此，FMDV 2A序列为改造整个蛋白质或结构域提供了机会，使得它们能够高效地共翻译切割开。
We describe the construction of a plasmid (pCAT2AGUS) encoding a polyprotein in which a 19 amino acid sequence spanning the 2A region of the foot-and-mouth disease virus (FMDV) polyprotein was inserted between the reporter genes chloramphenicol acetyl transferase (CAT) and beta-glucuronidase (GUS) maintaining a single, long open reading frame. Analysis of translation reactions programmed by this construct showed that the inserted FMDV sequence functioned in a manner similar to that observed in FMDV polyprotein processing: the CAT2AGUS polyprotein underwent a cotranslational, apparently autoproteolytic, cleavage yielding CAT-2A and GUS. Analysis of translation products derived from a series of constructs in which sequences were progressively deleted from the N-terminal region of the FMDV 2A insertion showed that cleavage required a minimum of 13 residues. The FMDV 2A sequence therefore provides the opportunity to engineer either whole proteins or domains such that they are cleaved apart cotranslationally with high efficiency.