Structure-based engineering and comparison of novel split inteins for protein ligation

A. Sesilja Aranko, Jesper S. Oeemig, Dongwen Zhou, Tommi Kajander, Alexander Wlodawer, Hideo Iwaï*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

39 Citations (Scopus)


Protein splicing is an autocatalytic process involving self-excision of an internal protein domain, the intein, and concomitant ligation of the two flanking sequences, the exteins, with a peptide bond. Protein splicing can also take place in trans by naturally split inteins or artificially split inteins, ligating the exteins on two different polypeptide chains into one polypeptide chain. Protein trans-splicing could work in foreign contexts by replacing the native extein sequences with other protein sequences. Protein ligation using protein trans-splicing increasingly becomes a useful tool for biotechnological applications such as semi-synthesis of proteins, segmental isotopic labeling, and in vivo protein engineering. However, only a few split inteins have been successfully applied for protein ligation. Naturally split inteins have been widely used, but they are cross-reactive to each other, limiting their applications to multiple-fragment ligation. Based on the three-dimensional structures including two newly determined intein structures, we derived 21 new split inteins from four highly efficient cis-splicing inteins, in order to develop novel split inteins suitable for protein ligation. We systematically compared trans-splicing of 24 split inteins and tested the cross-activities among them to identify orthogonal split intein fragments that could be used in chemical biology and biotechnological applications.

Original languageEnglish
Pages (from-to)1023-1034
Number of pages12
JournalMolecular BioSystems
Issue number5
Publication statusPublished - 2014
MoE publication typeA1 Journal article-refereed


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