Fig. 8 |. Split intein-based strategies for generating semisynthetic proteins.

a, Proteins can be site-specifically modified at their termini by splitting them into two separate pieces, each fused to a split-intein fragment. For N-terminal modification, a short, synthetic N-terminal segment of the protein of interest (POI) (POI^N) is fused to the N-terminal split intein fragment (Int^N), whereas the recombinantly expressed C-terminal POI fragment (POI^C) is fused to the C-terminal split-intein fragment (Int^C). Upon association and protein trans-splicing (PTS), an N-terminally modified POI is produced. Similarly, C-terminal modification is performed by making the Int^C–POI^C complex synthetically. To modify a central segment of a POI (POI^Cen), orthogonal intein pairs are used in a tandem protein splicing scheme in which the POI^Cen is synthetic and fused to intein fragments at both termini. Int^N₁ and Int^C₁ as well as Int^N₂ and Int^C₂ represent two pairs of orthogonal split inteins. b, Transpeptidase-assisted intein ligation (TAIL) enables N-terminal (N-tail) and C-terminal (C-tail) modification of proteins by combining sortase- and intein-mediated protein ligation. Split inteins are further split into small overhangs (Int^N overhang and Int^C overhang) containing sortase recognition sequences LAYTG or LAATG and truncated, inactive inteins. The reversible, sortase-mediated transpeptidation step generates active split inteins carrying synthetic cargo, which associate with their split-intein partner for protein semisynthesis through irreversible PTS. Note that sortase and the truncated Int^N are fused in N-tail to increase the reaction rate and thereby suppress premature cleavage of Int^C–POI^N complex.