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).
- Evans, T. et al. (1998) Protein Sci. 7, 2256-2264. PMID: 9827992
- Dawson, P. et al. (1994) Science 266, 776-779. PMID: 7973629
- Tam, J. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 12485-12489. PMID: 8618926
- Muir, T. et al. (1998) Proc. Natl. Acad. Sci. USA 95,6705-6710. PMID: 9618476
- Severinov, K. and Muir, T. (1998) J. Biol. Chem. 273, 16205-16209. PMID: 9632677
FAQs for Peptide Ligation
- What are the causes of inefficient ligation?
- What is the protocol for IPL?
- What cleavage reagent should be used for IPL? What is the efficiency of cleavage and IPL if I use MESNA or DTT?
- Which products should be ordered for IPL?
- Which residues at the C-terminus of the target protein may inhibit cleavage or cause in vivo cleavage when the pTXB vectors are used?
- What vectors are suitable for IPL and cyclization?
- How can the C-terminal fusion vectors be used to label the C-terminus of the target protein?
- How should I store the eluted protein with a C-terminal thioester if I do not want to do ligation immediately?
- Do you have some references on IPL?
- What is Intein-mediated Protein Ligation (IPL)?
- What has IPL been used for?
- Where can find all IMPACT FAQs?
Protocols for Peptide Ligation
- 5 Minute Transformation Protocol (C2527)
- 5 Minute Transformation Protocol (C2528)
- 5 Minute Transformation Protocol (C2529)
- 5 Minute Transformation Protocol (C2566)
- 5 Minute Transformation Protocol (C3010)
- 5 Minute Transformation Protocol (C3013)
- 5 Minute Transformation Protocol (C3016)
- Affinity Purification and On-column Cleavage (E6901)
- Construction of the Fusion Plasmid (E6901)
- Expression Using SHuffle®
- Fusion Constructs (E6901)
- Fusion Protein Expression (E6901)
- High Efficiency Transformation Protocol
- High Efficiency Transformation Protocol (C2529)
- High Efficiency Transformation Protocol (C2566)
- High Efficiency Transformation Protocol (C3013)
- High Efficiency Transformation Protocol (C3016)
- Preparation of Media and Solutions (E6901)
- Primer Design for Restriction Enzyme Cloning (E6901)
- Protein Expression Using BL21(DE3) (C2527)
- Protein Expression Using Lemo21(DE3) (C2528)
- Protein Expression Using NiCo21(DE3) (C2529)
- Protein Expression with T7 Strains
- Protocol for Expression Using T7 Express (C2566)
- Protocol for Expression Using T7 Express Iq (C3016)
- Protocol for Expression Using T7 Express lysY (C3010)
- Protocol for Expression Using T7 Express lysY/Iq (C3013)
- Protocol for Removal of IMAC Contaminating Proteins (C2529)
- Simplified Expression and Purification Protocol (E6901)
- Transformation Protocol (C2528)
- Transformation Protocol for BL21(DE3) Competent Cells (C2527)
Application Notes Peptide Ligation
Bypassing Common Obstacles in Protein Expression
Competent Cells Brochure
The Competent Cells brochure provides information on the different competent cell strains for cloning and protein expression available from NEB.
Protein Expression & Purification Brochure
The Protein Expression and Purification brochure provides information on the advanced tools for protein expression and purification offered by NEB.
- IMPACT™ Vectors and Applications