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Protein splicing is a form of posttranslational processing that consists of the excision of an intervening polypeptide sequence, the intein, from a protein, accompanied by the concomitant joining of the flanking polypeptide sequences, the exteins, by a peptide bond. It requires neither cofactors nor auxiliary enzymes and involves a series of four intramolecular reactions, the first three of which occur at a single catalytic center of the intein. Protein splicing can be modulated by mutation and converted to highly specific self-cleavage and protein ligation reactions that are useful protein engineering tools. Some of the reactions characteristic of protein splicing also occur in other forms of protein autoprocessing, ranging from peptide bond cleavage to conjugation with nonprotein moieties. These mechanistic similarities may be the result of convergent evolution, but in at least one case-hedgehog protein autoprocessing-there is definitely a close evolutionary relationship to protein splicing.

Protein splicing involves the excision of an internal protein segment, the intein, from a precursor protein and the concomitant ligation of the flanking N-and Cterminal regions. It occurs in mesophilic bacteria, yeast, and thermophilic archaea. The ability to control protein splicing of a thermophilic intein by temperature and pH in a foreign protein context facilitated the study of the mechanism of protein splicing in thermophiles. On the other hand, no direct studies have been done on the mechanism of protein splicing in mesophiles. We examined the splicing of a chimeric protein containing the intein of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiae that involves cysteines rather than serines at the reaction center. The steps in the splicing process were deduced by analyzing intermediates and side products that accumulated as a result of amino acid substitutions and were found to be analogous to those occurring in thermophiles. Moreover, appropriate amino acid replacements allowed us to develop the first mesophilic in vitro protein splicing system as well as strategies for modulating the rate of protein splicing and for converting the splicing reaction to an efficient protein cleavage reaction at either splice junction.Protein splicing is a novel mode of gene expression that has been described in mesophilic bacteria and yeast and in extremely thermophilic archaea (1-6). It is a process in which a single gene directs the synthesis of two separate proteins by the precise excision of an internal protein segment, the intein, from a precursor protein and the concomitant ligation of the flanking N-and C-terminal regions, the exteins, to yield two new proteins (7). Some of the excised inteins are homing endonucleases that catalyze lateral transfer of their DNA coding sequences by an intein homing mechanism (8 -11), whereas the ligated exteins are usually enzymes with a specific cellular function.Efficient protein splicing also occurs when inteins are transferred into heterologous proteins, suggesting that all structural and catalytic elements needed for the splicing reaction reside in the inteins plus the first C-extein residue (4,(12)(13)(14). The protein splicing function of inteins is independent of their homing endonuclease activity (15) and depends on highly conserved amino acid residues at both splice junctions. A hydroxyl-or thiol-containing residue (Ser, Thr, or Cys) is always present at the positions that immediately follow the two splice junctions, and the sequence His-Asn is invariant at the intein C terminus. Substitution of any of these conserved residues retards or abolishes protein splicing (2,12,13,15).The fact that the thermophilic archaeal inteins in a foreign protein context undergo efficient splicing only at elevated temperatures (25-65°C) opened the way for the development of an in vitro system to study the mechanism of protein splicing (14). We constructed a fusion protein containing the intein from the thermostable DNA polymerase of Pyroccocus sp. GB-D, which could be expressed i...

Protein splicing involves the excision of an internal domain from a precursor protein and the ligation of the external domains so as to generate two new proteins. Study of this process has recently been facilitated by the isolation of a precursor and a branched intermediate from a thermophilic protein splicing element expressed in a foreign protein context. Two aspects of protein splicing are examined in this paper. We demonstrate a succinimide at the C‐terminus of the spliced internal protein, implicating cyclization of asparagine in resolution of the branched intermediate, and we identify an alkali‐labile bond in the branched intermediate. A revised protein splicing model based on these experimental results is presented.

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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...

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Preparation of bispecific antibodies by the chemical reassociation of monovalent fragments derived from monoclonal mouse immunoglobulin G1 is inefficient because of side reactions during reoxidation of the multiple disulfide bonds linking the heavy chains. These side reactions can be avoided by using specific dithiol complexing agents such as arsenite and effecting disulfide formation with a thiol activating agent such as 5,5'-dithiobis(2-nitrobenzoic acid). In this way bispecific antibodies were obtained in high yield and free of monospecific contaminants from monoclonal mouse immunoglobulin G1 fragments. The bispecific antibodies were used as agents for the selective immobilization of enzymes.