ArticleThe Journal of biological chemistry2011
Highly efficient and more general cis- and trans-splicing inteins through sequential directed evolution.
Article in The Journal of biological chemistry, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 50 papers.
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Who cites it
50 citing papers in PubMed, 102 citations in OpenAlex.
- Regulation of N-terminal cleavage and splicing in mini-Ter DnaE-3 intein by E23G and R50Q mutations.Biotechnology letters · 2026Article
- Generating Ultra-Fast Protein trans-Splicing of a Cysteine-Less and Semisynthetic Split Intein for Chemical Protein Labeling.Chembiochem : a European journal of chemical biology · 2026Article
- An enhanced split intein-mediated ligation (SIML) platform for rapid discovery and functional screening of circular bacteriocins.Frontiers in bioengineering and biotechnology · 2026Article
- Three-Segment Protein Labeling Using a Highly Efficient and Cysteine-Less Split Intein Identified with Computational Prediction of Aggregation Properties.Angewandte Chemie (International ed. in English) · 2025Article
- Split RNA switch orchestrates pre- and post-translational control to enable cell type-specific gene expression.Nature communications · 2025Article
- Optogenetics with Atomic Precision─A Comprehensive Review of Optical Control of Protein Function through Genetic Code Expansion.Chemical reviews · 2025Review
- Ligation of multiple protein domains using orthogonal inteins with non-native splice junctions.Protein science : a publication of the Protein Society · 2024Article
- Article
- Engineered mRNA-ribosome fusions for facile biosynthesis of selenoproteins.Proceedings of the National Academy of Sciences of the United States of America · 2024Article
- Efficient splicing of the CPE intein derived from directed evolution of theActa biochimica et biophysica Sinica · 2023Article
- Extein residues regulate the catalytic function of Spl DnaX intein enzyme by restricting the near-attack conformations of the active-site residues.Protein science : a publication of the Protein Society · 2023Article
- Nature-inspired protein ligation and its applications.Nature reviews. Chemistry · 2023Review
- Dual incorporation of non-canonical amino acids enables production of post-translationally modified selenoproteins.Frontiers in molecular biosciences · 2023Article
- Creating Selenocysteine-Specific Reporters Using Inteins.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Intein-based Design Expands Diversity of Selenocysteine Reporters.Journal of molecular biology · 2022Article
- Using selenocysteine-specific reporters to screen for efficient tRNAMethods in enzymology · 2022Article
- Functional cross-talk between phosphorylation and disease-causing mutations in the cardiac sodium channel NaProceedings of the National Academy of Sciences of the United States of America · 2021Article
- An alternative domain-swapped structure of the Pyrococcus horikoshii PolII mini-intein.Scientific reports · 2021Article
- A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction.Nature communications · 2021Article
- Greening the synthesis of peptide therapeutics: an industrial perspective.RSC advances · 2020Review
Corrections and comments
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Authors and funding
6 authors at 2 institutions in 2 countries.
Funding
Abstract
Inteins are internal protein sequences that post-translationally self-excise and splice together the flanking sequences, the so-called exteins. Natural and engineered inteins have been used in many practical applications. However, inteins are often inefficient or inactive when placed in a non-native host protein and may require the presence of several amino acid residues of the native exteins, which will then remain as a potential scar in the spliced protein. Thus, more general inteins that overcome these limitations are highly desirable. Here we report sequential directed evolution as a new approach to produce inteins with such properties. Random mutants of the Ssp (Synechocystis sp. PCC 6803) DnaB mini-intein were inserted into the protein conferring kanamycin resistance at a site where the parent intein was inactive for splicing. The mutants selected for splicing activity were further improved by iterating the procedure for two more cycles at different positions in the same protein. The resulting improved inteins showed high activity in the positions of the first rounds of selection, in multiple new insertion sites, and in different proteins. One of these inteins, the M86 mutant, which accumulated 8 amino acid substitutions, was also biochemically characterized in an artificially split form with a chemically synthesized N-terminal intein fragment consisting of 11 amino acids. When compared with the unevolved split intein, it exhibited an ∼60-fold increased rate in the protein trans-splicing reaction and a K(d) value for the interaction of the split intein fragments improved by an order of magnitude. Implications on the intein structure-function, practical application, and evolution are discussed.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.