Protein splicing is a post-translational process by which an intervening polypeptide, the intein, excises itself from the flanking polypeptides, the exteins, coupled to ligation of the exteins. The Ion protease of Pyrococcus abyssi (Pab) is interrupted by an intein. When over-expressed as a fusion protein in Escherichia colt, the Pab Ion protease intein can promote efficient protein splicing. Mutations that block individual steps of splicing generally do not lead to unproductive side reactions, suggesting that the intein tightly coordinates the splicing process. The intein can splice, although it has Lys in place of the highly conserved penultimate His, and mutants of the intein in the C-terminal region lead to the accumulation of stable branched-ester intermediate. (C) 2010 Elsevier Inc. All rights reserved.