An expanded library of orthogonal split inteins enables modular multi-peptide assemblies

Filipe Pinto, Ella Lucille Thornton, Baojun Wang

Research output: Contribution to journalArticlepeer-review


Inteins are protein segments capable of joining adjacent residues via a peptide bond. In this process known as protein splicing, the intein itself is not present in the final sequence, thus achieving scarless peptide ligation. Here, we assess the splicing activity of 34 inteins (both uncharacterized and known) using a rapid split fluorescent reporter characterization platform, and establish a library of 15 mutually orthogonal split inteins for in vivo applications, 10 of which can be simultaneously used in vitro. We show that orthogonal split inteins can be coupled to multiple split transcription factors to implement complex logic circuits in living organisms, and that they can also be used for the in vitro seamless assembly of large repetitive proteins with biotechnological relevance. Our work demonstrates the versatility and vast potential of an expanded library of orthogonal split inteins for their use in the fields of synthetic biology and protein engineering.
Original languageEnglish
Article number1529
Pages (from-to)1-15
Number of pages15
JournalNature Communications
Publication statusPublished - 23 Mar 2020


  • synthetic biology
  • proteins
  • split intein
  • post translational modification

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