Evans T C, Martin D, Kolly R, Panne D, Sun L, Ghosh I, Chen L, Benner J, Liu X Q, Xu M Q
New England Biolabs, Inc., Beverly, Massachusetts 01915, USA.
J Biol Chem. 2000 Mar 31;275(13):9091-4. doi: 10.1074/jbc.275.13.9091.
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 trans-splicing 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 N- and 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.
来自集胞藻属PCC6803（Ssp DnaE内含肽）的dnaE基因的一种天然存在的分裂内含肽已被证明能在体外和体内的外源蛋白环境中高效介导反式剪接。一种顺式剪接Ssp DnaE内含肽构建体表现出与反式剪接形式相似的剪接活性，这表明N端和C端内含肽片段具有高亲和力相互作用。开发了一种体外反式剪接系统，该系统使用细菌表达的Ssp DnaE内含肽的N端片段以及细菌表达或化学合成的内含肽C端片段。与人工分裂内含肽不同，Ssp DnaE内含肽片段可以在天然条件下在体外重组，以介导剪接以及肽键裂解。这一特性使得开发一种柱上反式剪接系统成为可能，该系统能够方便地分离反应物和产物。此外，Ssp DnaE内含肽的反式剪接活性已成功应用于体内蛋白质的环化。同样，在几丁质树脂上分离未剪接的前体使得环化反应能够在体外进行。因此，Ssp DnaE内含肽是体内和体外蛋白质操作中一种潜在的重要蛋白质。
