Mutant inteins

Muir et al. (1998) realized that the intein reaction could be used to facilitate a native chemical ligation with a synthetic N-terminal cysteine-containing peptide or cysteine-containing molecule. With the discovery of a mutant intein that could form an intermediate thioester but not go on to complete the splice and ligation reaction (Xu and Perler, 1996 Chong et al.,... 

Fusion protein containing the expressed protein, the mutant intein, and the CBD... 

Figure 17.29 An expressed protein containing a mutant intein segment can undergo self cleavage to form an N-terminal cysteine residue, which then can be reacted with a thioester probe to label specifically the protein via an amide bond.

As noted above, the production of recombinant protein thioesters was first achieved by the use of mutant inteins rendered incapable of resolving their... 

Figure 8.8 Expressed protein ligation. The final step of protein splicing by the intein is inactivated by the mutation of the C-tenninal Asn to Ala. Proteins expressed as in-frame N-tenninal fusions to such mutant inteins can be cleaved by thiols to give corresponding protein (N-peptide) thioester derivatives. The tagged inteins are removed. The N-peptide thioesters can then react with an aCys-containing peptide (C-peptide)...

Fusion vectors are available that combine a recombinant protein with a mutant mini intein segment (not containing an endonuclease domain) and followed by a chitin binding domain (CBD Zhang et al., 2001). These mutants typically also have an alanine substitution that replaces the cysteine or serine/threonine usually found on the C-extein splice junction. Alanine... 

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... 

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