Protein splicing involves the self-catalyzed excision of protein splicing elements, or inteins, from f lanking polypeptide sequences, or exteins, leading to the formation of new proteins in which the exteins are linked directly by a peptide bond. To study the enzymology of this interesting process we have expressed and purified N-and C-terminal segments of the Mycobacterium tuberculosis RecA intein, each Ϸ100 amino acids long, fused to appropriate exteins. These fragments were reconstituted into a functional protein splicing element by renaturation from 6 M urea. When renaturation was carried out in the absence of thiols, the reconstituted splicing element accumulated as an inactive disulfide-linked complex of the two intein fragments, which could be induced to undergo protein splicing by reduction of the disulfide bond. This provided a useful tool for separately investigating the requirements for the reconstitution of the intein fragments to yield a functional protein splicing element and for the protein splicing process per se. For example, the pH dependence of these processes was quite different, with reconstitution being most efficient at pH 8.5 and splicing most rapid at pH 7.0. The availability of such an in vitro protein splicing system opens the way for the exploration of intein structure and the unusual enzymology of protein splicing. In addition, this trans-splicing system is a potential protein ligase that can link any two polypeptides fused to the N-and C-terminal intein segments.Protein splicing is an unusual process by which the flow of information from a gene to its protein product is modulated posttranslationally so as to yield two functionally unrelated proteins. It involves the precise, self-catalyzed excision of an intervening polypeptide sequence, the intein, from an inactive precursor protein with the concomitant joining of the flanking sequences, the exteins, to produce a new functional protein (Fig. 1). All information and catalytic groups required for protein splicing reside in the intein and the two flanking amino acids. With the elucidation of the chemical mechanism of protein splicing (for review see refs. 1 and 2), it has become clear that inteins constitute a class of highly unusual enzymes: (i) they catalyze three mechanistically distinct reactions, two of which, acyl rearrangement of a peptide bond adjacent to cysteine or serine (3, 4) and cyclization of asparagine coupled to peptide bond cleavage (5, 6), have also been found to occur naturally in polypeptides, but only under extreme conditions or at very slow rates; (ii) they act on amino acid residues at their own N and C termini, so that the intein enzymes are also their own substrate, analogous to the role of catalytic RNA in the self-splicing of group I introns (7); and (iii) they catalyze a transesterification reaction between their N and C termini, and their catalytic center therefore comprises both extremities of the polypeptide chain, a situation that is rarely encountered in conventional enzymes and suggests an un...

95, 3543-3548).We have replaced the C-terminal intein fragment of this system with synthetic peptides comprising 35-50 of the C-terminal residues of the RecA intein. The N-terminal intein fragment and the synthetic peptide were reconstituted by renaturation from guanidinium chloride. In the absence of added reductants, a disulfide-linked dimer of the N-terminal fragment and the peptide accumulated and could be induced to splice by reduction of its disulfide bond. The intermediate and spliced products were identified by polyacrylamide gel electrophoresis, mass spectrometry, and derivatization with thiol-reactive biotin followed by Western blotting with a streptavidin-enzyme conjugate. This is the first example of protein splicing involving a synthetic intein fragment and opens the way for studying the active site structure and function of the intein by the use of different synthetic peptides, including ones with non-natural amino acids.Protein splicing is a mechanism for the post-translational processing of proteins that involves the self-catalyzed excision of an intervening polypeptide, the intein, and the subsequent formation of a new protein by joining the flanking sequences, the exteins, by a peptide bond. It involves the catalysis of three mechanistically unrelated reactions at a single catalytic center, which resides entirely within the intein (for reviews, see Refs.1 and 2). The intein can thus be viewed as an exceedingly complex enzyme, and the investigation of the catalytic mechanisms involved in protein splicing is of great interest.With the aim of obtaining an in vitro protein splicing system whose structure and function can be examined by biochemical and biophysical methods, we are developing a minimal protein splicing element from the 440-residue RecA intein of Mycobacterium tuberculosis by eliminating the portions of the intein that are not essential for protein splicing. Most inteins are interrupted by homing endonuclease domains, which account for about one-half of the intein sequence but can be deleted without eliminating protein splicing ability (3-5). In addition, we found that the RecA intein can be split into two fragments that can complement each other so as to promote trans-splicing (5). This made possible the development of an in vitro transsplicing system composed of 105-residue N-and C-terminal fragments of the M. tuberculosis RecA intein (6). An in vitro trans-splicing system based on the Pol-1 intein of the hyperthermophilic archeon, Pyrococcus sp. GB-D, was recently described (7). The results described in this paper further advance our approach by replacing the natural C-terminal intein fragment with 35-50-residue synthetic polypeptides. The resulting semisynthetic protein splicing element was able to catalyze protein splicing with high efficiency. This exciting development will facilitate the study of the structure and catalytic function of the C-terminal portion of the intein by replacing specific residues with other natural amino acids or with unnatural amino acids and structural p...

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Protein splicing in trans results in the ligation of two protein or peptide segments linked to appropriate intein fragments. We have characterized the trans‐splicing reaction mediated by a naturally expressed, approximately 100‐residue N‐terminal fragment of the Mycobacterium tuberculosis intein and a synthetic peptide containing the 38 C‐terminal intein residues, and found that the splicing reaction was very versatile and robust. The efficiency of splicing was nearly independent of temperature between 4 and 37°C and pH between 6.0 and 7.5, with only a slight decline at pH values as high as 8.5. In addition, there was considerable flexibility in the choice of the C‐terminal intein fragment, no significant difference in protein ligation efficiency being observed between reactions utilizing the N‐terminal fragment and either the naturally expressed 107‐residue C‐terminal portion of the intein, much smaller synthetic peptides, or the 107‐residue C‐terminal intein fragment modified by fusion of a maltose binding protein domain to its N‐terminus. The ability to use different types of the C‐terminal intein fragments and a broad range of reaction conditions make protein splicing in trans a versatile tool for protein ligation. © 2000 John Wiley & Sons, Inc. Biopoly 51: 355–362, 1999