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 NiCo21(DE3) (C2529)
- Expression Using SHuffle®
- Fusion Constructs (E6901)
- Fusion Protein Expression (E6901)
- High Efficiency Transformation Protocol
- High Efficiency Transformation Protocol (C2529)
- High Efficiency Transformation Protocol (C3013)
- High Efficiency Transformation Protocol (C3016)
- High Efficiency Transformation Protocol (C2566)
- 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 with T7 Strains
- Protocol for Expression Using T7 Express (C2566)
- Protocol for Expression Using T7 Express Iq (C3016)
- Protocol for Expression Using T7 Express lys/Iq(C3013)
- Protocol for Expression Using T7 Express lysY (C3010)
- 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)
- E. coli Lemo21(DE3)
- Intein-Mediated Protein Ligation (IPL) and Labeling with the IMPACT™ Kit
- Protein Expression with T7 Express Strains
- Transformation of SHuffle® Competent Cell Strains
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.
Bypassing Common Obstacles in Protein Expression
- IMPACT™ Vectors and Applications
Other Tools & Resources
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