protein splicing


Summary: The excision of in-frame internal protein sequences (INTEINS) of a precursor protein, coupled with ligation of the flanking sequences (EXTEINS). Protein splicing is an autocatalytic reaction and results in the production of two proteins from a single primary translation product: the intein and the mature protein.

Top Publications

  1. Mujika J, Lopez X, Mulholland A. Mechanism of C-terminal intein cleavage in protein splicing from QM/MM molecular dynamics simulations. Org Biomol Chem. 2012;10:1207-18 pubmed publisher
    b>Protein splicing is a post-translational process in which a biologically inactive protein is activated by the release of a segment denoted as an intein. The process involves four steps...
  2. Sonntag T, Mootz H. An intein-cassette integration approach used for the generation of a split TEV protease activated by conditional protein splicing. Mol Biosyst. 2011;7:2031-9 pubmed publisher
    Ligand-induced conditional protein splicing (CPS) using a split intein allows the covalent reconstitution of a protein from two polypeptide fragments...
  3. Mizutani R, Nogami S, Kawasaki M, Ohya Y, Anraku Y, Satow Y. Protein-splicing reaction via a thiazolidine intermediate: crystal structure of the VMA1-derived endonuclease bearing the N and C-terminal propeptides. J Mol Biol. 2002;316:919-29 pubmed
    b>Protein splicing excises an internal intein segment from a protein precursor precisely, and concomitantly ligates flanking N and C-extein polypeptides at the respective sides of the precursor...
  4. Muralidharan V, Muir T. Protein ligation: an enabling technology for the biophysical analysis of proteins. Nat Methods. 2006;3:429-38 pubmed
  5. Flavell R, Muir T. Expressed protein ligation (EPL) in the study of signal transduction, ion conduction, and chromatin biology. Acc Chem Res. 2009;42:107-16 pubmed publisher
    ..These techniques and concepts are applicable to many other systems, and ongoing advances promise to extend this semisynthetic technique to increasingly complex biological problems. ..
  6. Starokadomskiĭ P. [Protein splicing]. Mol Biol (Mosk). 2007;41:314-30 pubmed
    b>Protein splicing is a post-translational autocatalytic excision of internal protein sequence (intein) with the subsequent ligation of the flanking polypeptides (exteins)...
  7. Kerrigan A, Powers T, Dorval D, Reitter J, Mills K. Protein splicing of the three Pyrococcus abyssi ribonucleotide reductase inteins. Biochem Biophys Res Commun. 2009;387:153-7 pubmed publisher
    ..its own excision from the exteins, concomitant with the ligation of the exteins, in a process called protein splicing. The alpha subunit of the ribonucleotide reductase of the extreme thermophile Pyrococcus abyssi is ..
  8. Ding Y, Xu M, Ghosh I, Chen X, Ferrandon S, Lesage G, et al. Crystal structure of a mini-intein reveals a conserved catalytic module involved in side chain cyclization of asparagine during protein splicing. J Biol Chem. 2003;278:39133-42 pubmed
    ..Our structural modeling suggests that this catalytic module is conserved in the C-terminal subdomains of inteins from diverse organisms. ..
  9. Ludwig C, Pfeiff M, Linne U, Mootz H. Ligation of a synthetic peptide to the N terminus of a recombinant protein using semisynthetic protein trans-splicing. Angew Chem Int Ed Engl. 2006;45:5218-21 pubmed

More Information


  1. Mootz H, Muir T. Protein splicing triggered by a small molecule. J Am Chem Soc. 2002;124:9044-5 pubmed
    ..In principle, this can be achieved by harnessing protein splicing, a posttranslational editing process that results in the precise removal of an internal domain (termed an ..
  2. Volkmann G, Sun W, Liu X. Controllable protein cleavages through intein fragment complementation. Protein Sci. 2009;18:2393-402 pubmed publisher
    ..These findings significantly expand the effectiveness of intein-based protein cleavages, and they also reveal important insights of intein structural flexibility and fragment complementation. ..
  3. Allemand E, Dokudovskaya S, Bordonné R, Tazi J. A conserved Drosophila transportin-serine/arginine-rich (SR) protein permits nuclear import of Drosophila SR protein splicing factors and their antagonist repressor splicing factor 1. Mol Biol Cell. 2002;13:2436-47 pubmed
    ..Together, the results imply that the mechanism by which SR proteins are imported to the nucleus is conserved between Drosophila and humans. ..
  4. Lew B, Paulus H. An in vivo screening system against protein splicing useful for the isolation of non-splicing mutants or inhibitors of the RecA intein of Mycobacterium tuberculosis. Gene. 2002;282:169-77 pubmed
    b>Protein splicing involves the self-catalyzed excision of an intervening sequence, the intein, from a precursor protein, with the concomitant ligation of the flanking extein sequences to yield a new polypeptide...
  5. Tori K, Dassa B, Johnson M, Southworth M, Brace L, Ishino Y, et al. Splicing of the mycobacteriophage Bethlehem DnaB intein: identification of a new mechanistic class of inteins that contain an obligate block F nucleophile. J Biol Chem. 2010;285:2515-26 pubmed publisher
    ..The Cys or Ser at the N terminus of most inteins initiates a four-step protein splicing reaction by forming a (thio)ester bond at the N-terminal splice junction...
  6. Du Z, Zheng Y, Patterson M, Liu Y, Wang C. pK(a) coupling at the intein active site: implications for the coordination mechanism of protein splicing with a conserved aspartate. J Am Chem Soc. 2011;133:10275-82 pubmed publisher
    b>Protein splicing is a robust multistep posttranslational process catalyzed by inteins. In the Mtu RecA intein, a conserved block-F aspartate (D422) coordinates different steps in protein splicing, but the precise mechanism is unclear...
  7. Apgar J, Ross M, Zuo X, Dohle S, Sturtevant D, Shen B, et al. A predictive model of intein insertion site for use in the engineering of molecular switches. PLoS ONE. 2012;7:e37355 pubmed publisher
    ..These findings can be used to enrich the selection of insertion sites capable of supporting intein splicing and hosting an intein switch. ..
  8. Minato Y, Ueda T, Machiyama A, Shimada I, Iwai H. Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing. J Biomol NMR. 2012;53:191-207 pubmed publisher
    ..Both approaches remain to be further developed for facilitating preparations of segmental isotope-labelled samples without extensive optimizations for ligation. ..
  9. Lacerda Pinheiro S, Jegat N, Septier D, Priam F, Bonnefoix M, Bitard J, et al. Early in vivo and in vitro effects of amelogenin gene splice products on pulp cells. Eur J Oral Sci. 2006;114 Suppl 1:232-8; discussion 254-6, 381-2 pubmed
  10. Cheriyan M, Pedamallu C, Tori K, Perler F. Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues. J Biol Chem. 2013;288:6202-11 pubmed publisher
    Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond...
  11. Kimura R, Tran A, Camarero J. Biosynthesis of the cyclotide Kalata B1 by using protein splicing. Angew Chem Int Ed Engl. 2006;45:973-6 pubmed
  12. Kanno A, Ozawa T, Umezawa Y. Intein-mediated reporter gene assay for detecting protein-protein interactions in living mammalian cells. Anal Chem. 2006;78:556-60 pubmed we developed a new reporter gene assay for detecting cytosolic protein-protein interactions based on protein splicing of transcription factors with DnaE inteins...
  13. Dassa B, Amitai G, Caspi J, Schueler Furman O, Pietrokovski S. Trans protein splicing of cyanobacterial split inteins in endogenous and exogenous combinations. Biochemistry. 2007;46:322-30 pubmed
    ..excise from protein precursors and ligate their flanking regions with a peptide bond, in a process called protein splicing. Intein-containing DNA polymerases of cyanobacteria and nanoarchaea are naturally split into two separate ..
  14. Iwai H, Züger S, Jin J, Tam P. Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme. FEBS Lett. 2006;580:1853-8 pubmed
    ..b>Protein splicing with the Npu DnaEN is also more tolerant of amino acid substitutions in the C-terminal extein sequence.
  15. Skretas G, Wood D. Regulation of protein activity with small-molecule-controlled inteins. Protein Sci. 2005;14:523-32 pubmed
  16. Buskirk A, Ong Y, Gartner Z, Liu D. Directed evolution of ligand dependence: small-molecule-activated protein splicing. Proc Natl Acad Sci U S A. 2004;101:10505-10 pubmed
    ..To restore activity in a ligand-dependent manner, we linked protein splicing to cell survival or fluorescence in Saccharomyces cerevisiae...
  17. Mills K, Manning J, Garcia A, Wuerdeman L. Protein splicing of a Pyrococcus abyssi intein with a C-terminal glutamine. J Biol Chem. 2004;279:20685-91 pubmed
    b>Protein splicing involves the excision of an intervening polypeptide sequence, the intein, from a precursor protein and the concomitant ligation of the flanking polypeptides, the exteins, by a peptide bond...
  18. Amitai G, Dassa B, Pietrokovski S. Protein splicing of inteins with atypical glutamine and aspartate C-terminal residues. J Biol Chem. 2004;279:3121-31 pubmed
    ..The observed C-terminal cleavage in the Chy intein ending with Ala cannot be explained by cyclization of this residue. We present and discuss several new models for reactions in the protein-splicing pathway. ..
  19. Nichols N, Benner J, Martin D, Evans T. Zinc ion effects on individual Ssp DnaE intein splicing steps: regulating pathway progression. Biochemistry. 2003;42:5301-11 pubmed
    ..By inhibiting both the splicing pathway and spontaneous hydrolysis with Zn(2+), reactants can be diverted from the trans splicing to the trans cleavage pathway where DTT and EDTA can regulate N- and C-terminal cleavage, respectively. ..
  20. Posey K, Gimble F. Insertion of a reversible redox switch into a rare-cutting DNA endonuclease. Biochemistry. 2002;41:2184-90 pubmed
    ..These studies demonstrate that homing endonuclease activity can be controlled using a molecular switch. ..
  21. Chen L, Pradhan S, Evans T. Herbicide resistance from a divided EPSPS protein: the split Synechocystis DnaE intein as an in vivo affinity domain. Gene. 2001;263:39-48 pubmed
  22. Wu H, Hu Z, Liu X. Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803. Proc Natl Acad Sci U S A. 1998;95:9226-31 pubmed
    ..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. ..
  23. Perler F, Xu M, Paulus H. Protein splicing and autoproteolysis mechanisms. Curr Opin Chem Biol. 1997;1:292-9 pubmed
    ..However, numerous examples of self-catalyzed protein rearrangements have recently been discovered, including protein splicing and autoproteolysis of hedgehog proteins, glycosylasparaginases and pyruvoyl enzyme precursors...
  24. Liu D, Xu R, Cowburn D. Segmental isotopic labeling of proteins for nuclear magnetic resonance. Methods Enzymol. 2009;462:151-75 pubmed publisher
    ..We also describe the protocol used in our lab for the segmental isotopic labeling of a 50-kDa protein Csk (C-terminal Src kinase) using expressed protein ligation methods. ..
  25. Poulter R, Goodwin T, Butler M. The nuclear-encoded inteins of fungi. Fungal Genet Biol. 2007;44:153-79 pubmed
    ..The diverse fungal inteins provide a resource for biotechnology using their protein splicing or homing endonuclease capabilities.
  26. Bezerra W, Carvalho C, Moreira R, Grangeiro T. Establishment of a heterologous system for the expression of Canavalia brasiliensis lectin: a model for the study of protein splicing. Genet Mol Res. 2006;5:216-23 pubmed
    During its biosynthesis in developing Canavalia brasiliensis seeds, the lectin ConBr undergoes a form of protein splicing in which the order of the N- and C-domains of the protein is reversed...
  27. Mills K, Perler F. The mechanism of intein-mediated protein splicing: variations on a theme. Protein Pept Lett. 2005;12:751-5 pubmed
    Intein-mediated protein splicing is facilitated by four separate but coordinated nucleophilic displacement reactions that result in the excision of the intein and the ligation of the flanking polypeptides, called the exteins...
  28. Sun P, Ye S, Ferrandon S, Evans T, Xu M, Rao Z. Crystal structures of an intein from the split dnaE gene of Synechocystis sp. PCC6803 reveal the catalytic model without the penultimate histidine and the mechanism of zinc ion inhibition of protein splicing. J Mol Biol. 2005;353:1093-105 pubmed
    ..chelating the highly conserved Cys160 and Asp140 reveals the structural basis of Zn2+-mediated inhibition of protein splicing. Furthermore, it is of interest to observe that the carbonyl carbon atom of Asn159 and N(eta) of Arg73 are 2...
  29. Anraku Y, Mizutani R, Satow Y. Protein splicing: its discovery and structural insight into novel chemical mechanisms. IUBMB Life. 2005;57:563-74 pubmed
    b>Protein splicing is a posttranslational cellular process, in which an intervening protein sequence (intein) is self-catalytically excised out from a nascent protein precursor and the two flanking sequences (N- and C-exteins) are ligated ..
  30. Shi J, Muir T. Development of a tandem protein trans-splicing system based on native and engineered split inteins. J Am Chem Soc. 2005;127:6198-206 pubmed
    ..The technology should provide a convenient approach to the segmental isotopic or fluorogenic labeling of specific domains within the context of large multidomain proteins. ..
  31. Perler F. Protein splicing of inteins and hedgehog autoproteolysis: structure, function, and evolution. Cell. 1998;92:1-4 pubmed
  32. Gorbalenya A. Non-canonical inteins. Nucleic Acids Res. 1998;26:1741-8 pubmed
    Previous analyses have shown that inteins (protein splicing elements) employ two structural organizations: the 'canonical' Nintein-Dod-inteinC found in dozens of inteins and a 'non-canonical' Nintein-inteinC described in two inteins, ..
  33. Cheriyan M, Perler F. Protein splicing: A versatile tool for drug discovery. Adv Drug Deliv Rev. 2009;61:899-907 pubmed publisher
    ..Moreover, recent advances in small molecule triggered protein splicing allow for tunable post-translational control of protein function in vivo...
  34. Sharma S, Zhang A, Wang H, Harcum S, Chong S. Study of protein splicing and intein-mediated peptide bond cleavage under high-cell-density conditions. Biotechnol Prog. 2003;19:1085-90 pubmed
    b>Protein splicing elements (inteins), capable of catalyzing controllable peptide bond cleavage reactions, have been used to separate recombinant proteins from affinity tags during affinity purification...
  35. Hanada K, Yewdell J, Yang J. Immune recognition of a human renal cancer antigen through post-translational protein splicing. Nature. 2004;427:252-6 pubmed
    ..human leukocyte antigen-A3 MHC class I molecules presenting a nine-residue FGF-5 peptide generated by protein splicing. This process, previously described strictly in plants and unicellular organisms, entails post-translational ..
  36. Aranko A, Züger S, Buchinger E, Iwai H. In vivo and in vitro protein ligation by naturally occurring and engineered split DnaE inteins. PLoS ONE. 2009;4:e5185 pubmed publisher
    ..Based on kinetic analysis of the protein splicing reactions, we propose a general strategy to improve ligation yields by protein trans-splicing, which could ..
  37. Li J, Sun W, Wang B, Xiao X, Liu X. Protein trans-splicing as a means for viral vector-mediated in vivo gene therapy. Hum Gene Ther. 2008;19:958-64 pubmed publisher
    Inteins catalyze protein splicing in a fashion similar to how self-splicing introns catalyze RNA splicing. Splitinteins catalyze precise ligation of two separate polypeptides through trans-splicing in a highly specific manner...
  38. Davies M, Barraclough D, Stewart C, Joyce K, Eccles R, Barraclough R, et al. Expression and splicing of the unfolded protein response gene XBP-1 are significantly associated with clinical outcome of endocrine-treated breast cancer. Int J Cancer. 2008;123:85-8 pubmed publisher
  39. Tyszkiewicz A, Muir T. Activation of protein splicing with light in yeast. Nat Methods. 2008;5:303-5 pubmed publisher
    ..Here we report a genetically encoded system for controlling a post-translational process, protein splicing, with light...
  40. Mills K, Dorval D, Lewandowski K. Kinetic analysis of the individual steps of protein splicing for the Pyrococcus abyssi PolII intein. J Biol Chem. 2005;280:2714-20 pubmed
    b>Protein splicing involves the excision of an intervening polypeptide, the intein, from flanking polypeptides, the exteins, concomitant with the specific ligation of the exteins...
  41. Schwartz E, Saez L, Young M, Muir T. Post-translational enzyme activation in an animal via optimized conditional protein splicing. Nat Chem Biol. 2007;3:50-4 pubmed
    ..We used an optimized conditional protein splicing system to rapidly trigger the in vivo ligation of two inactive fragments of firefly luciferase in a tunable ..
  42. Sun W, Yang J, Liu X. Synthetic two-piece and three-piece split inteins for protein trans-splicing. J Biol Chem. 2004;279:35281-6 pubmed
    ..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. ..
  43. Perler F, Olsen G, Adam E. Compilation and analysis of intein sequences. Nucleic Acids Res. 1997;25:1087-93 pubmed
    We have compiled a list of all the inteins (protein splicing elements) whose sequences have been published or were available from on-line sequence databases as of September 18, 1996...
  44. Pearl E, Tyndall J, Poulter R, Wilbanks S. Sequence requirements for splicing by the Cne PRP8 intein. FEBS Lett. 2007;581:3000-4 pubmed publisher
    The dependence of protein splicing on conserved residues of the Cne PRP8 intein was assessed by alanine scanning mutagenesis in a foreign protein context...
  45. Gogarten J, Senejani A, Zhaxybayeva O, Olendzenski L, Hilario E. Inteins: structure, function, and evolution. Annu Rev Microbiol. 2002;56:263-87 pubmed
    ..In particular, this review considers inteins as selfish or parasitic genetic elements, a point of view that explains many otherwise puzzling aspects of inteins. ..
  46. Buchinger E, Aachmann F, Aranko A, Valla S, Skjak Braek G, Iwai H, et al. Use of protein trans-splicing to produce active and segmentally (2)H, (15)N labeled mannuronan C5-epimerase AlgE4. Protein Sci. 2010;19:1534-43 pubmed publisher
    ..The activity of the ligated AlgE4 was verified by two different enzyme activity assays, demonstrating that ligated AlgE4 displays the same catalytic activity as wild-type AlgE4. ..
  47. Liu X. Protein-splicing intein: Genetic mobility, origin, and evolution. Annu Rev Genet. 2000;34:61-76 pubmed
    ..A self-splicing intein catalyzes its own removal from the host protein through a posttranslational process of protein splicing. A mobile intein displays a site-specific endonuclease activity that confers genetic mobility to the intein ..
  48. Ozawa T, Nishitani K, Sako Y, Umezawa Y. A high-throughput screening of genes that encode proteins transported into the endoplasmic reticulum in mammalian cells. Nucleic Acids Res. 2005;33:e34 pubmed based on the reconstitution of two split fragments of enhanced green fluorescent protein (EGFP) by protein splicing with DnaE from Synechocystis PCC6803...
  49. Telenti A, Southworth M, Alcaide F, Daugelat S, Jacobs W, Perler F. The Mycobacterium xenopi GyrA protein splicing element: characterization of a minimal intein. J Bacteriol. 1997;179:6378-82 pubmed insertion in the gyrA gene of Mycobacterium xenopi is the smallest known naturally occurring active protein splicing element (intein). Comparison with other mycobacterial gyrA inteins suggests that the M...
  50. Carvajal Vallejos P, Pallissé R, Mootz H, Schmidt S. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J Biol Chem. 2012;287:28686-96 pubmed publisher
    Inteins excise themselves out of precursor proteins by the protein splicing reaction and have emerged as valuable protein engineering tools in numerous and diverse biotechnological applications...
  51. Dalgaard J, Moser M, Hughey R, Mian I. Statistical modeling, phylogenetic analysis and structure prediction of a protein splicing domain common to inteins and hedgehog proteins. J Comput Biol. 1997;4:193-214 pubmed
    ..Here, a specific and sensitive hidden Markov model (HMM) of protein splicing domain shared by inteins and the hedgehog proteins has been trained and employed for further analysis...
  52. Ozawa T, Takeuchi T, Kaihara A, Sato M, Umezawa Y. Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time. Anal Chem. 2001;73:5866-74 pubmed
    ..protein-protein interactions based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing (Ozawa, T.; et al. Anal. Chem. 2000, 72, 5151-5157...
  53. Paulus H. Inteins as enzymes. Bioorg Chem. 2001;29:119-29 pubmed
    ..However, as our understanding of protein autoprocessing reactions such as protein splicing advances, it is becoming clear that they have many of the hallmarks of enzymatic reactions...