Protein trans -splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803

Abstract

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...

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

“…In nature, a split intein could arise from a contiguous intein as a result of genomic rearrangements breaking the intein coding sequence. Considering the large number of naturally occurring split inteins (14,23,24), why do they all have a single and common split site? Because we found multiple new split sites compatible with trans-splicing, we can now exclude the possibility that this common split site of natural split inteins is the only site compatible with protein trans-splicing.…”

“…An interesting event of intein evolution is the loss of sequence continuity in some inteins, which apparently produced the DnaE split intein that exists in two fragments and is capable of protein trans-splicing (23). A pair of split DnaE genes produces two precursor polypeptides, with one consisting of the N-terminal part of DnaE (N-extein) followed by the N-terminal part of intein (N-intein) and another consisting of the C-terminal part of DnaE (C-extein) preceded by the Cterminal part of the intein (C-intein).…”

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].…”

Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli

“…We have pursued intracellular backbone cyclization to generate bio-stable peptide libraries. This procedure, termed SICLOPPS 19,20 , utilizes the Synechocystis sp PCC6803 DnaE split intein 21 . By rearranging the order of the elements of the intein, an active cis-intein (I C :target peptide:I N ) is yielded that upon splicing results in cyclization of the target protein/peptide sequence (Fig.…”