Ligation intein-mediated protein

Fig. 3. Intein-mediated protein ligation. The IMPACT system allows affinity purification of proteins fused to an intein-CBD tag and their further isolation with a C-terminal thioester moiety (A), or an N-terminal cysteine (B). (A), N-terminal intein splicing for thioester isolation. Target protein (protein 1) is expressed in E. coli with C-terminally located intein-CBD tag. After specific binding to the chitin resin, the thiol reagent provokes the cleavage of the peptide bond between the target protein and the intein. Whereas the intein-CBD tag remains bound to the chitin resin, the protein thioester is eluted from the column. (B), C-terminal cleavage to obtain N-terminally...

S. Nock, Establishment of intein-mediated protein ligation under denaturing conditions C-terminal labeling of a single-chain antibody for biochip screening, Bioconjugate Chem. 2002, 13, 707-712. 

Fig. 1.4 A I ntein-mediated protein ligation (IPL) (also called expressed protein ligation EPL). B The chemical mechanism of protein cyclization by the IPL/EPL approach. HAC denotes the sequence of the active site of the intein, e.g. His-Ala-Cys. CBD stands for chitin-binding domain.

Otomo T, Ito N, Kyogoku Y, Yamazaki T. NMR observation of selected segments in a larger protein central-segment isotope labeling through intein-mediated ligation. Biochemistry 1999 38 16040-16044. 

Wood Rl, Pascoe DD, Brown ZK, Medlicott EM, Kriek M, Neylon C, Peter L, Roach PL (2004) Optimized conjugation of a fluorescent label to proteins via intein-mediated activation and ligation. Bioconjugate Chem 15 366-372... 

C-terminal peptide a-thioester, mUdly activated peptide ester acting as a valuable key intermediate for the synthesis/semi-synthesis of polypeptides and proteins by both chemical ligation and the Aimoto thioester approach. The synthesis of the peptide a-thioester (—r thioester) can be performed by standard SPPS using Boc- or Fmoc-based chemistry or, for larger target polypeptides, by application of intein-based bacterial expression systems. Peptide a-thioester synthesis can also be carried out based on an N-S acyl shift reaction mediated by a thiol ligation auxiliary [F. B. Perler, E. Adams, Curr. Opin. Biotechnol. 2000, 377 D. Swinnen, D. Hilvert, Org. Lett. 2000, 2, 2439 R. Quaderer, D. Hilvert, Org. Lett. 2001, 3, 3181 T. W. Muir, Annu. Rev. Biochem. 2003, 72, 249 T. Kawakami et al.. Tetrahedron Lett. 2005, 46, 8805 J. A. Camarero, A. R. Mitchell, Prot. Pept. Lett. 2005, 12, 723]. 

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