Fusion intein domain

Figure 7.2 Expressed protein ligation (ERL) requires a protein C-terminal thioester that can be obtained by empioying pTWIN vectors bearing an intein domain and a chitinbinding domain (CBD). The fusion protein...

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... 

Figure 4.8 The intein system as used for fusion proteins (Xu, 2000). The fusion proteins undergo self-cleavage at the upstream splice junction. The amino acids that participate directly are shown, and the remainder of the intein and target protein are drawn as boxes. Usually, the intein is conjugated to a chitin-binding domain, so purification of the fusion protein can occur by using a chitin-conjugated column. Self-cleavage can occur overnight.

Chen et al. (2008) have expressed a codon-optimized form of the CM4 peptide in E. coli. In this case, two alternative expression/purification systems were examined namely the widely used glutathione-S-transferase (GST) and a chitin-binding domain (CBD) system with associated intein splicing (New England Biolabs Inc.). The GST system failed to allow recovery of expressed fusion protein. In contrast, the CBD/intein system allowed the recovery of 110 mg/L fusion protein with a final RP-HPLC purification step yielding 2.1 mg/L of pure peptide which displayed antimicrobial activity against E. coli Ki2D3i and Salmonella. 

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