Fig. 11. Selected protein ligation approaches with potential for large-scale production of therapeutic peptides. (A) In Native Chemical Ligation a peptide thioester reacts with a second peptide bearing an N-terminal Cys residue through trans-thioesterification and subsequent S-to-N-acyl shift. Potential sources for peptide thioester generation include (i) hydrazides,^165,166 (ii) N-acylureas,^139,140 (iii) bis(2-sulfanylethyl)amido (SEA) amides,^141,167 (iv) peptides fused with intein proteins,^145,168 and (v) peptides with C-terminal enzymatic recognition motifs or tags.^146,147,169 (B) Protein trans-splicing involves peptides fused with split intein sequences that self-assemble to form an active intein unit. The intein is then autocatalytically excised while ligating the peptides of interest. PTS requires a Cys or Ser residue N-terminal on the first split intein partner (Int^N), typically an Asn residue C-terminal on the second split intein partner (Int^C) which cyclises to a succinimide moiety upon excision, and a Cys, Ser, or Thr residue N-terminal on the C-extein peptide.^123,124 (C) Transpeptidase and peptidyl ligase enzymes catalyse amide bond formation between peptides (chemoenzymatic transamidation/ligation), each with their own peptide recognition motifs and requirement for efficient catalysis.