Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein

Abstract

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...

Synthetic Two-piece and Three-piece Split Inteins for Protein trans-Splicing

“…Previously known split inteins, naturally occurring or artificial, all have a similar split site corresponding to or inside the endonuclease domain. They have shown variability and limitations in solubility, reaction kinetics, and compatibility with certain extein sequences (20,(25)(26)(27)(28)30). New split sites identified in this study that produced intein fragments of various lengths and interacting ␤-strands can be useful for comparative studies of split intein properties such as fragment binding and reaction kinetics; they can also expand the toolbox of protein trans-splicing in biotechnology.…”

“…However, it may also suggest that splitting at any other site is incompatible with protein trans-splicing and therefore not tolerated, which can be examined by splitting inteins at other sites followed by testing for possible trans-splicing. Synthetic two-piece split inteins have been engineered before in laboratories by splitting the coding sequences of contiguous inteins, but their split sites corresponded with those of the naturally occurring split inteins (25)(26)(27). Both naturally occurring and synthetic split inteins have found many practical uses, which include producing trans-spliced recombinant proteins and circularized proteins or peptides for various purposes (28 -30).…”

Solution structure of DnaE intein from Nostoc punctiforme: Structural basis for the design of a new split intein suitable for site‐specific chemical modification

“…Protein splicing is a self-catalytic reaction catalyzed by an intervening sequence in a host protein, termed an intein, which ligates the flanking protein sequences, termed exteins, by a peptide bond and concomitantly excises itself from the host protein [3]. Protein splicing in trans could ligate two foreign peptide chains that are fused with either N-or C-terminal fragments (N-or C-inteins) of a split intein [4][5][6]. Protein trans-splicing system has opened many applications including segmental isotopic labelling of proteins, protein cyclization, in vivo protein engineering, and site-specific chemical modifications [6][7][8][9][10][11][12][13][14][15].…”

Mycobacterium tuberculosis RecA Intein Possesses a Novel ATP-dependent Site-specific Double-stranded DNA Endonuclease Activity

“…The deduced amino acid sequence of M. tuberculosis recA intervening sequence disclosed the existence of two copies of LAGLIDADG motifs. The protein-splicing ability of PI-MtuI 1 has been studied extensively (22)(23)(24)(25); however, the endonuclease activity has not been demonstrated. To this end, PI-MtuI was cloned, overexpressed, and purified, to explore its biochemical functions.…”