Highly efficient and more general cis- and trans-splicing inteins through sequential directed evolution

Chemoenzymatic approaches bear potential advantages over chemical ligation methods in the generation of site-selectively modified proteins. Two polypeptide segments, of which one is synthetically and the other recombinantly produced, can be linked through protein trans-splicing to give a semisynthetic protein (see Figure). The Ssp DnaB intein was previously shown to support protein trans-splicing when split following aa 11 (S1-site).¹ Taking advantage of the short Int^N segment it was established as an attractive tool for ligating synthetic peptide fragments to the N terminus of proteins.^2,3

We here report the biochemical characterization of an evolved mutant of the Ssp DnaB intein.⁴ The trans-splicing activity of the mutant was investigated in the context of a model protein and compared to the parent intein. Strikingly, the first-order rate of trans-splicing was increased ~60-fold, such that about 90% splice product formation is achieved within 30 min with negligible formation of the C-terminal cleavage by-product. Additionally, the evolved mutant showed high splicing activity with a non-native extein residue at the (-1) position, for which the parent intein was almost inactive.⁴

Thus, even though the mutations were selected in the context of a cis-intein under cellular expression conditions in E. coli, they also dramatically improved the splicing capability of the artificially split intein under in vitro conditions.