A split intein capable of protein transsplicing is identified in a DnaE protein of the cyanobacterium Synechocystis sp. strain PCC6803. The N-and C-terminal halves of DnaE (catalytic subunit ␣ of DNA polymerase III) are encoded by two separate genes, dnaE-n and dnaE-c, respectively. These two genes are located 745,226 bp apart in the genome and on opposite DNA strands. The dnaE-n product consists of a N-extein sequence followed by a 123-aa intein sequence, whereas the dnaE-c product consists of a 36-aa intein sequence followed by a C-extein sequence. The N-and C-extein sequences together reconstitute a complete DnaE sequence that is interrupted by the intein sequences inside the ␤-and -binding domains. The two intein sequences together reconstitute a split mini-intein that not only has intein-like sequence features but also exhibited protein trans-splicing activity when tested in Escherichia coli cells.Inteins have been defined as protein sequences embedded in-frame within a precursor protein sequence and excised during a maturation process termed protein splicing (1, 2). Protein splicing is a post-translational event involving precise excision of the intein sequence and concomitant ligation of the flanking sequences (N-and C-exteins) by a normal peptide bond (3-5). Most reported inteins are thought to be bifunctional elements, possessing a protein splicing activity and an endonuclease activity (6-9). Crystal structure of the Sce VMA1 intein revealed a two-domain structure, with domain I consisting of the N-and C-terminal regions of the intein sequence and domain II formed by the middle part of the intein sequence (10). Domain I (or a part of it) was suggested to be the splicing domain, whereas domain II corresponded to the endonuclease domain. Such a bipartite structure may be applicable to many other inteins, as has been suggested by studies including mutagenesis (11, 12) and sequence statistical modeling (7-9). Functional studies of mini-inteins, either found in nature or engineered in vitro, also confirmed such a two-domain model (13-15), further suggesting that the N-and C-terminal regions of an intein make up a functional splicing domain. Molecular mechanisms of protein splicing involve an N3S (or N3O) acyl shift at the N-terminal splice site (16-18), formation of a branched intermediate (19,20), and cyclization of an invariant Asn residue at the C terminus of intein to form succinimide (21), leading to excision of the intein. The ligated exteins undergo an S3N (or O3N) acyl shift to form a native peptide bond (21). Amino acid residues that are implicated in the splicing mechanism include a nucleophilic amino acid (Cys, Ser, or Thr) both at the beginning of the intein sequence and at the beginning of the C-extein sequence, an internal His, and a His-Asn dipeptide at the end of the intein sequence. In crystal structures of two inteins, these amino acids are indeed positioned at or near the active site of protein splicing (10,22).Approximately 50 intein-coding sequences have been found in Ͼ20 differe...

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...

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Spiders store spidroins in their silk glands as high concentration aqueous solutions, spinning these dopes into fibres with outstanding mechanical properties. Aciniform (or wrapping) silk is the toughest spider silk and is devoid of the short amino acid sequence motifs characteristic of the other spidroins. Using solution-state NMR spectroscopy, we demonstrate that the 200 amino acid Argiope trifasciata AcSp1 repeat unit contrasts with previously characterized spidroins, adopting a globular 5-helix bundle flanked by intrinsically disordered N- and C-terminal tails. Split-intein-mediated segmental NMR-active isotope-enrichment allowed unambiguous demonstration of modular and malleable “beads-on-a-string” concatemeric behaviour. Concatemers form fibres upon manual drawing with silk-like morphology and mechanical properties, alongside secondary structuring and orientation consistent with native AcSp1 fibres. AcSp1 structural stability varies locally, with the fifth helix denaturing most readily. The structural transition of aciniform spidroin from a mostly α-helical dope to a mixed α-helix/β-sheet-containing fibre can be directly related to spidroin architecture and stability.

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