Peptide Ligation

The IPL reaction allows the ligation of a synthetic peptide or a protein with an N-terminal cysteine residue to the thioester on the C-terminus of an expressed protein through a native peptide bond (1). The IPL protocol employs the IMPACT™ (NEB #E6901) C-terminal fusion vectors to express and purify a protein of interest and to generate a thioester at its C-terminus. For IPL we recommend the use of pTXB1 when possible. The IPL reaction applies the chemistry described for "native chemical ligation" which fuses two synthetic peptides when the N-terminal cysteine of one peptide attacks a C-terminal thioester of another peptide (2,3). Initially, a new thioester bond is formed by transthioesterification involving attack by the sulfhydryl group of the N-terminal cysteine residue on the C-terminal thioester. The transitory ligation product then undergoes a spontaneous S-N acyl rearrangement from a thioester to a stable peptide bond. This technique has also been described as "expressed protein ligation" (4,5).

1. Evans, T. et al. (1998) Protein Sci. 7, 2256-2264. PMID: 9827992 
2. Dawson, P. et al. (1994) Science 266, 776-779. PMID: 7973629 
3. Tam, J. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 12485-12489. PMID: 8618926 
4. Muir, T. et al. (1998) Proc. Natl. Acad. Sci. USA 95,6705-6710. PMID: 9618476 
5. Severinov, K. and Muir, T. (1998) J. Biol. Chem. 273, 16205-16209. PMID: 9632677