An artificially split class 3 intein.

Inteins excise themselves from precursor polypeptides through protein splicing, joining N- and C-exteins with a peptide bond. Split inteins are expressed as separate polypeptides that undergo protein trans splicing (PTS). Here, we demonstrate PTS can be achieved using an artificially split class 3 intein. Because class 3 inteins use an internal initiating nucleophile near the C-extein junction, rather than the first residue of the intein, both catalytic nucleophiles are present on a single polypeptide. This results in a compact arrangement of catalytic nucleophiles for PTS compared to the standard arrangement for split class 1 inteins.