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

Abstract

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...

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.…”

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 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]. However, protein ligation by protein trans-splicing can be significantly modulated by the junction sequences as well as by the extein sequences, which could limit the applications of protein trans-splicing [16].…”

Crystal Structure of a Mini-intein Reveals a Conserved Catalytic Module Involved in Side Chain Cyclization of Asparagine during Protein Splicing

“…Serine or lysine may substitute for histidine in hydrogen bonding to an oxyanion. The Methanococcus jannaschii phosphoenolpyruvate synthase intein, representing those inteins lacking a penultimate histidine, could undergo splicing due to the presence of the Asn in block F. Indeed the Ssp DnaE intein, which lacks histidine in the penultimate position but has an aspartate in block F, is splicing-proficient (42)(43)(44).…”