The IPL reaction allows the ligation of a synthetic peptide or a protein with an N-terminal cysteine residue to the C-terminus of a bacterially expressed protein through a native peptide bond [Evans et al., (1998) Protein Sci.7, 2256-2264]. The IPL protocol employs the IMPACT 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 [Dawson et al.,(1994)Science266, 776-779; Tam et al., (1995) Proc. Natl. Acad. Sci. USA92, 12485-12489]. 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" [Muir et al.,(1998)Proc. Natl. Acad. Sci. USA95,6705-6710; Severinov and Muir(1998)J. Biol. Chem.273, 16205-16209].