The lon‐b protease of the extreme thermophile Pyrococcus abyssi is interrupted by an intein. Inteins are intervening polypeptides that catalyze their own excision from, and the concurrent ligation of, the flanking polypeptides, called exteins. We have shown that the P. abyssi lon‐b protease intein can facilitate protein splicing when over‐expressed in E. coli as a fusion protein with affinity domains serving as exteins. We have generated inteins with site directed mutations of the nucleophilic residues at the splice junctions, and these inteins cannot facilitate splicing. In other inteins, such mutations lead to cleavage at the splice junctions, but these side reactions are not as significant with the P. abyssi lon intein. We have also made mutations to study the influence of the penultimate Lys on splicing and/or C‐terminal side reactions. This Lys replaces a highly conserved His. We are also studying the influence of Gly residues in the extein that flank the intein insertion site. This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.
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