Protein splicing in cis and trans. (a) Protein splicing in cis. An internal protein (Intein, Int) is spliced out from a precursor protein, concomitantly ligating the two flanking proteins (N-extein and C-extein). ExN and ExC indicate N-extein and C-extein, respectively. (b) Canonical intein with homing endonuclease domain (EN), Mini-intein without EN, and split intein. (c) A cartoon model of NpuDnaE intein [20,21]. The natural split-site (C35) and engineered split-site (C15) are marked with arrows [22]. The N-terminal split fragment of the natural split intein is colored in blue. IntN used in this study is colored in blue or cyan. The region corresponding to the IntC fragment used in this study is highlighted in green. (d) Protein ligation by PTS using a split intein. PTS ligates the N-terminal fragment of a protein of interest (POIN) fused with the N-terminal split intein (IntN) and the C-terminal fragment of POI (POIC) fused with the C-terminal split intein (IntC). PTS results in ligated POIN-POIC and excised IntN and IntC. The figures were adapted from [5].