Protein splicing involves the excision of an intervening polypeptide sequence, the intein, from a precursor protein and the concomitant ligation of the flanking polypeptides, the exteins, by a peptide bond. Most reported inteins have a C-terminal asparagine residue, and it has been shown that cyclization of this residue is coupled to peptide bond cleavage between the intein and C-extein. We show that the intein interrupting the DNA polymerase II DP2 subunit in Pyrococcus abyssi, which has a C-terminal glutamine, is capable of facilitating protein splicing. Substitution of an asparagine for the C-terminal glutamine moderately improves the rate and extent of protein splicing. However, substitution of an alanine for the penultimate histidine residue, with either asparagine or glutamine in the C-terminal position, prevents protein splicing and facilitates cleavage at the intein N terminus. The intein facilitates in vitro protein splicing only at temperatures above 30°C and can be purified as a nonspliced precursor. This temperature dependence has enabled us to characterize the optimal in vitro splicing conditions and determine the rate constants for splicing as a function of temperature.Protein splicing is a post-translational auto-processing event by which an intervening polypeptide sequence, the intein, facilitates both its own excision from the flanking polypeptides, or exteins, as well as the ligation of the these segments (reviewed in Ref. 1). All of the catalytic groups required for protein splicing have been shown to lie within the intein and the two flanking amino acids (1).The splicing mechanism of the majority of inteins has been described in detail (1). The first step of splicing involves an N-O or N-S acyl rearrangement of the peptide bond at the intein N terminus to an ester or thioester (Fig. 1, step A). The second step is a transesterification between the nucleophilic residue at the N terminus of the C-extein 1 (Cys, Ser, or Thr) and the newly formed ester or thioester at the intein N terminus (Fig.
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