Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803
Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803
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DOI：
10.1074/jbc.275.13.9091
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发表时间：
2000-03-31
影响因子：
4.8
通讯作者：
Xu, MQ
中科院分区：
文献类型：
--
作者：
Evans, TC;Martin, D;Xu, MQ
来自集胞藻PCC 6803的dnaE基因的天然存在的分裂内含肽（Ssp DnaE内含肽）已显示在外源蛋白背景下介导有效的体内和体外反式剪接。顺式剪接的Ssp DnaE内含肽具有与反式剪接相似的剪接活性，表明N端和C端内含肽片段具有高亲和力的相互作用。开发了一种体外反式剪接系统，其使用细菌表达的Ssp DnaE内含肽的N-末端片段和细菌表达的或化学合成的内含肽C-末端片段。与人工分裂的内含肽不同，Ssp DnaE内含肽片段可以在天然条件下在体外重构以介导剪接以及肽键切割。这种性质允许开发柱上反式剪接系统，该系统允许反应物和产物的容易分离。此外，Ssp DnaE内含肽的反式剪接活性被成功地应用于蛋白质的体内环化，并且，未剪接的前体在几丁质树脂上的分离允许环化反应在体外进行。因此，Ssp DnaE内含肽代表了用于体内和体外蛋白质操纵的潜在重要蛋白质。
A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been shown to mediate efficient in vivo and in vitro transsplicing in a foreign protein context. A cis-splicing Ssp DnaE intein construct displayed splicing activity similar to the trans-splicing form, which suggests that the Nand C-terminal intein fragments have a high affinity interaction. An in vitro trans-splicing system was developed that used a bacterially expressed N-terminal fragment of the Ssp DnaE intein and either a bacterially expressed or chemically synthesized intein C-terminal fragment. Unlike artificially split inteins, the Ssp DnaE intein fragments could be reconstituted in vitro under native conditions to mediate splicing as well as peptide bond cleavage. This property allowed the development of an on-column trans-splicing system that permitted the facile separation of reactants and products. Furthermore, the trans-splicing activity of the Ssp DnaE intein was successfully applied to the cyclization of proteins in vivo, Also, the isolation of the unspliced precursor on chitin resin allowed the cyclization reaction to proceed in vitro. The Ssp DnaE intein thus represents a potentially important protein for in vivo and in vitro protein manipulation.