Synthetic two-piece and three-piece split inteins for protein trans-splicing
Synthetic two-piece and three-piece split inteins for protein trans-splicing
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DOI：
10.1074/jbc.m405491200
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发表时间：
2004-08-20
影响因子：
4.8
通讯作者：
Liu, XQ
中科院分区：
文献类型：
--
作者：
Sun, WC;Yang, J;Liu, XQ
内含肽是蛋白质介入序列，可以自我切除并同时将侧翼多肽拼接在一起。能够进行蛋白质转拼的两段式分裂内含肽已在自然界中发现并在实验室中进行了改造，但它们都具有与内含肽的核酸内切酶结构域相对应的相似分裂位点。内含肽可以在其他位点分裂并进行转剪吗？在测试了 13 个被改造为 Ssp DnaB 迷你内含肽的分裂位点后，我们报告了三个新的分裂位点的发现，每个位点产生了能够进行蛋白质转拼的两部分分裂内含肽。这三个功能性分裂位点位于内含肽结构的β链之间的不同环区域，其中一个距离内含肽开头仅11个氨基酸。由于不同的内含肽具有相似的结构和相似的β链，因此这些新的分裂位点可以推广到其他内含肽。我们还首次证明了三段式分裂内含肽可以在蛋白质转拼中发挥作用。这些发现对内含肽的结构-功能、进化和在生物技术中的应用具有重要意义。
Inteins are protein-intervening sequences that can self-excise and concomitantly splice together the flanking polypeptides. Two-piece split inteins capable of protein trans-splicing have been found in nature and engineered in laboratories, but they all have a similar split site corresponding to the endonuclease domain of the intein. Can inteins be split at other sites and do trans-splicing? After testing 13 split sites engineered into a Ssp DnaB mini-intein, we report the finding of three new split sites that each produced a two-piece split intein capable of protein trans-splicing. These three functional split sites are located in different loop regions between beta-strands of the intein structure, and one of them is just 11 amino acids from the beginning of the intein. Because different inteins have similar structures and similar beta-strands, these new split sites may be generalized to other inteins. We have also demonstrated for the first time that a three-piece split intein could function in protein trans-splicing. These findings have implications for intein structure-function, evolution, and uses in biotechnology.