Control of protein splicing by intein fragment reassembly

111

109

Inteins are protein-intervening sequences that can self-excise and concomitantly splice together the flanking polypeptides. Two-piece split inteins capable of protein trans-splicing have been found in nature and engineered in laboratories, but they all have a similar split site corresponding to the endonuclease domain of the intein. Can inteins be split at other sites and do transsplicing? After testing 13 split sites engineered into a Ssp DnaB mini-intein, we report the finding of three new split sites that each produced a two-piece split intein capable of protein trans-splicing. These three functional split sites are located in different loop regions between ␤-strands of the intein structure, and one of them is just 11 amino acids from the beginning of the intein. Because different inteins have similar structures and similar ␤-strands, these new split sites may be generalized to other inteins. We have also demonstrated for the first time that a three-piece split intein could function in protein trans-splicing. These findings have implications for intein structure-function, evolution, and uses in biotechnology.An intein is a protein-intervening sequence that catalyzes a protein-splicing reaction in which the intein sequence is precisely excised and its flanking sequences (N-and C-exteins) join with a peptide bond to produce the mature host protein (spliced protein) (1). The mechanism of protein splicing typically has four steps: two acyl rearrangements at the two splicing junctions, a trans-esterification between the two junctions, and a cyclization of the Asn residue at the C-terminal junction (2-4). Crystal structures of inteins revealed a splicing domain consisting of 11-12 ␤-strands and forming a compact horseshoe shape with the splicing junctions located in the central cleft (5-11). A majority of inteins also have a homing endonuclease domain inserted in the splicing domain sequence (12). These bifunctional inteins are ϳ350 -550 amino acids (aa) 1 long, although some extra large inteins are up to 1650 aa long and also contain tandem repeats (13,14). Nearly 200 intein and inteinlike sequences have been found in a wide variety of host proteins and in microorganisms belonging to bacteria, Archaea, and eukaryotes (12,15). Their sporadic phylogenetic distributions suggest lateral gene transfer through intein homing (16,17). Inteins generally share only low levels of sequence similarity, but they share striking similarities in structure, reaction mechanism, and evolution (4,18,19,21). It is thought that inteins first originated with just the splicing domain and then acquired the endonuclease domain, with the latter conferring genetic mobility to the intein. During intein evolution, however, some inteins lost their endonuclease domain to become mini-inteins consisting of just the ϳ130-aa protein-splicing domain plus a linker sequence of various lengths in place of the endonuclease domain (12,22).An interesting event of intein evolution is the loss of sequence continuity in some inteins, which apparently produced ...

Section: Discussionmentioning

confidence: 90%

mentioning

confidence: 99%

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

Sun

Yang

Liu

2004

111

109

“…The Ssp DnaE intein may be the intein of choice for in vitro trans-splicing experiments as it has been demonstrated to undergo the trans-splicing reaction without the need for a denaturation/renaturation step, as was necessary with other inteins (21,23,24). Also, the addition of the CBD to either the N-or C-terminal intein fragment had no detectable inhibitory effect on splicing.…”

Section: Discussionmentioning

confidence: 99%

“…In addition to inteins engineered to trans-splice (21)(22)(23)(24), a naturally occurring split intein was recently identified in the dnaE gene encoding the catalytic subunit of DNA polymerase III of Synechocystis sp. PCC6803 (25).…”

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confidence: 99%

Protein trans-Splicing and Cyclization by a Naturally Split Intein from the dnaE Gene ofSynechocystis Species PCC6803

Evans¹,

Martin²,

Kolly³

et al. 2000

186

177

A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been shown to mediate efficient in vivo and in vitro transsplicing in a foreign protein context. A cis-splicing Ssp DnaE intein construct displayed splicing activity similar to the trans-splicing form, which suggests that the Nand C-terminal intein fragments have a high affinity interaction. An in vitro trans-splicing system was developed that used a bacterially expressed N-terminal fragment of the Ssp DnaE intein and either a bacterially expressed or chemically synthesized intein C-terminal fragment. Unlike artificially split inteins, the Ssp DnaE intein fragments could be reconstituted in vitro under native conditions to mediate splicing as well as peptide bond cleavage. This property allowed the development of an on-column trans-splicing system that permitted the facile separation of reactants and products. Furthermore, the trans-splicing activity of the Ssp DnaE intein was successfully applied to the cyclization of proteins in vivo. Also, the isolation of the unspliced precursor on chitin resin allowed the cyclization reaction to proceed in vitro. The Ssp DnaE intein thus represents a potentially important protein for in vivo and in vitro protein manipulation.Protein splicing elements, termed inteins (1), catalyze their own excision from a primary translation product with the concomitant ligation of the flanking protein sequences (reviewed in Refs. 2-4). Inteins catalyze three highly coordinated reactions at the N-and C-terminal splice junctions (5, 6): 1) an acyl rearrangement at the N-terminal cysteine or serine; 2) a transesterification reaction between the two termini to form a branched ester or thioester intermediate; and 3) peptide bond cleavage coupled to cyclization of the intein C-terminal asparagine to free the intein. Inteins have been engineered to be versatile tools in protein purification (7-13), protein ligation (9, 10, 12, 14 -18), and in the formation of cyclic proteins and peptides (11,19,20). However, the ligation and cyclization approaches were limited by the need to generate an N-terminal cysteine and/or C-terminal thioester intermediate in vitro.In addition to inteins engineered to trans-splice (21-24), a naturally occurring split intein was recently identified in the dnaE gene encoding the catalytic subunit of DNA polymerase III of Synechocystis sp. PCC6803 (25). The N-terminal half of DnaE, followed by a 123-amino acid intein sequence, and the C-terminal half, preceded by a 36-amino acid intein sequence, are encoded by two open reading frames located more than 745 kilobases apart in the genome. When co-expressed in Escherichia coli, the two DnaE-intein fragments exhibited protein trans-splicing (25). In this report we have further investigated the cis-and trans-splicing activities of the Ssp DnaE intein in a foreign protein context. Furthermore, novel methods were developed that allow the on-column ligation of protein fragments as well as the in vivo and in vitro cyclization of p...

“…The in vitro biochemical study of protein splicing has been hampered by the fact that it is a self-catalyzed process that requires neither accessory proteins nor cofactors (2) and therefore proceeds rapidly under physiological conditions without the accumulation of intermediates. However, recent progress in the molecular dissection of inteins has made possible the expression of intein segments as fusion proteins that undergo protein splicing in trans after reconstitution in vitro (3,4). This advance as well as the recent discovery of a naturally occurring trans-splicing system (5, 6) has opened the way for the in vitro characterization of the protein-splicing process.…”

mentioning

confidence: 99%

Reversible Inhibition of Protein Splicing by Zinc Ion

Mills

Paulus

2001

Protein splicing involves the self-catalyzed excision of a protein-splicing element, the intein, from flanking polypeptides, the exteins, which are concomitantly joined by a peptide bond. Protein splicing involves the excision of an intervening polypeptide sequence, the intein, from a precursor protein and the concomitant joining of the flanking polypeptides, the exteins, by a peptide bond (see Ref. 1 for a recent review of protein splicing). The in vitro biochemical study of protein splicing has been hampered by the fact that it is a self-catalyzed process that requires neither accessory proteins nor cofactors (2) and therefore proceeds rapidly under physiological conditions without the accumulation of intermediates. However, recent progress in the molecular dissection of inteins has made possible the expression of intein segments as fusion proteins that undergo protein splicing in trans after reconstitution in vitro (3,4). This advance as well as the recent discovery of a naturally occurring trans-splicing system (5, 6) has opened the way for the in vitro characterization of the protein-splicing process.Protein splicing in trans requires the reassociation of an N-terminal and C-terminal fragment of an intein (N-intein and C-intein, respectively), each fused to an appropriate extein. Upon reassociation, the intein fragments form a functional protein-splicing active center, which mediates the formation of a peptide bond between the exteins, coupled to the excision of the N-and C-inteins (Fig. 1). In the experiments described in this paper, we used two different in vitro trans-splicing systems. The first was derived from the Mycobacterium tuberculosis RecA intein. It consisted of a 105-residue N-intein fused to Escherichia coli maltose-binding protein (MBP) 1 as the N-extein and a 107-residue C-intein fused to a polypeptide terminated with a His tag as the C-extein (4). The functional reassociation of the RecA intein segments requires prior denaturation and joint renaturation (4), even if the C-intein is replaced by a polypeptide as short as 35 amino acid residues (7). The second trans-splicing system was based on the naturally split DnaE intein of Synechocystis sp. PCC6803 (8), which has a 123-residue N-intein and a 36-residue C-intein and can undergo reassociation without prior denaturation (6). For the purpose of these studies, we fused the DnaE N-intein, joined to the 16 C-terminal residues of the natural N-extein, to E. coli MBP and a synthetic C-intein fused to the 5 N-terminal residues of the natural C-extein, followed by a His tag.A recent crystallographic study of a Saccharomyces cerevisiae VMA intein analog fused to short extein segments revealed that the intein contains a bound zinc ion (9). We decided to explore the effects of zinc ion on protein splicing using the experimental systems described above and discovered that zinc ion acts as a general and reversible inhibitor of protein splicing. No inhibitor of protein splicing had been available until now, and the reversible inhibition by Zn 2ϩ theref...