Inteins, expressed protein ligation using

Figure 17.14 An expressed protein containing a thioester intein tag that was subsequently modified by native chemical ligation to contain an alkyne group then can be labeled using an azido-fluorescein probe by the click chemistry reaction in the presence of Cu1+.

Figure 17.27 The EPL process involves a fusion protein containing an intein tag plus a CBD. The fusion protein is captured on an immobilized chitin resin and after removal of contaminating proteins, it is eluted using thiophenol, which cleaves at the thioester bond between the intein and the desired expressed protein. This releases a phenylth-ioester-activated protein that can be used in the native chemical ligation reaction with another peptide containing an N-terminal cysteine residue. Conjugation results in a native amide (peptide) bond formed between them.

Figure 17.28 EPL reactions can be used to couple a fusion protein to a surface containing a thioester derivative. After cells are grown and the fusion protein expressed, a pH and temperature shift causes intein cleavage with release of the expressed protein with an N-terminal cysteine residue. Reaction with the thioester surface results in a native chemical ligation reaction that forms an amide bond linkage with the expressed protein.

Evans, T. C. Jr., Benner, J., and Xu, M. Q. (1999) The in vitro ligation of bacteri-ally expressed proteins using an intein from Methanobacterium thermoautotroph-icum. J. Biol. Chem. 274, 3923-3926. 

EPL extends the applicability of native chemical ligation to recombinantly produced proteins using the mutant mini intein vector system. Proteins being expressed using this method will... 

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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