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Cell Oct 2010Protein splicing is a naturally occurring process in which a protein editor, called an intein, performs a molecular disappearing act by cutting itself out of a host...
Protein splicing is a naturally occurring process in which a protein editor, called an intein, performs a molecular disappearing act by cutting itself out of a host protein in a traceless manner. In the two decades since its discovery, protein splicing has been harnessed for the development of several protein-engineering methods. Collectively, these technologies help bridge the fields of chemistry and biology, allowing hitherto impossible manipulations of protein covalent structure. These tools and their application are the subject of this Primer.
Topics: Inteins; Protein Engineering; Protein Processing, Post-Translational; Protein Splicing; Recombinant Proteins
PubMed: 20946979
DOI: 10.1016/j.cell.2010.09.031 -
Nature Communications Oct 2019Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here...
Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here we present split selectable markers that each allow for selection of multiple "unlinked" transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR-Cas-mediated knock-ins. Split marker gene segments fused to protein splicing elements called "inteins" can be separately co-segregated with different transgenic vectors, and rejoin via protein trans-splicing to reconstitute a full-length marker protein in host cells receiving all intended vectors. Using a lentiviral system, we create and validate 2-split Hygromycin, Puromycin, Neomycin and Blasticidin resistance genes as well as mScarlet fluorescent proteins. By combining split points, we create 3- and 6-split Hygromycin resistance genes, demonstrating that higher-degree split markers can be generated by a "chaining" design. We adapt the split marker system for selecting biallelically engineered cells after CRISPR gene editing. Future engineering of split markers may allow selection of a higher number of genetic modifications in target cells.
Topics: CRISPR-Cas Systems; Cell Line, Tumor; Cinnamates; Drug Resistance, Bacterial; Gene Editing; Gene Transfer Techniques; Genetic Engineering; Genetic Vectors; HEK293 Cells; HeLa Cells; Humans; Hygromycin B; Induced Pluripotent Stem Cells; Inteins; Lentivirus; Luminescent Proteins; Neomycin; Nucleosides; Protein Splicing; Puromycin; Trans-Splicing; Transgenes
PubMed: 31672965
DOI: 10.1038/s41467-019-12891-2 -
Nature Chemistry Aug 2019Semisynthetic proteins engineered to contain non-coded elements such as post-translational modifications (PTMs) represent a powerful class of tools for interrogating...
Semisynthetic proteins engineered to contain non-coded elements such as post-translational modifications (PTMs) represent a powerful class of tools for interrogating biological processes. Here, we introduce a one-pot, chemoenzymatic method that allows broad access to chemically modified proteins. The approach involves a tandem transamidation reaction cascade that integrates intein-mediated protein splicing with enzyme-mediated peptide ligation. We show that this approach can be used to introduce PTMs and biochemical probes into a range of proteins including Cas9 nuclease and the transcriptional regulator MeCP2, which causes Rett syndrome when mutated. The versatility of the approach is further illustrated through the chemical tailoring of histone proteins within a native chromatin setting. We expect our approach will extend the scope of semisynthesis in protein engineering.
Topics: Amides; Humans; Methyl-CpG-Binding Protein 2; Protein Engineering; Protein Processing, Post-Translational; Protein Splicing
PubMed: 31263208
DOI: 10.1038/s41557-019-0281-2 -
The EMBO Journal Oct 1996Protein splicing results in the expression of two mature proteins from a single gene. After synthesis of a precursor protein, an internal segment (the intein) is excised...
Protein splicing results in the expression of two mature proteins from a single gene. After synthesis of a precursor protein, an internal segment (the intein) is excised and the external domains are joined together. A self-catalyzed mechanism for this cleavage-ligation reaction is presented, based on mutagenesis data and analysis of splicing intermediates. Mutations were used to block various steps in the protein splicing pathway, allowing each isolated step to be studied independently. A linear ester intermediate was identified and functional roles for the four conserved splice junction residues were determined. Understanding the mechanism of protein splicing provides a basis for protein engineering studies. For example, inteins can be constructed which fail to splice, but instead cleave the peptide bond at a chosen splice junction.
Topics: Bacterial Proteins; Carrier Proteins; Cysteine; DNA-Directed DNA Polymerase; Hydroxylamine; Hydroxylamines; Maltose-Binding Proteins; Models, Genetic; Mutagenesis, Insertional; Mutation; Protein Precursors; Protein Splicing; Recombinant Proteins; Tropomyosin
PubMed: 8895558
DOI: No ID Found -
Analytical Chemistry Dec 2001In this research, an improved detection system is described that allows an easy in vivo screening and selection of functional interactions between two interacting...
Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time.
In this research, an improved detection system is described that allows an easy in vivo screening and selection of functional interactions between two interacting proteins in bacteria. We earlier reported a new concept for detecting 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.): Two putative interacting proteins are genetically fused to the split VDE inteins, which are linked directly to the N- and C-terminal halves of the split EGFP. Association of the interacting proteins results in functional complementation of VDE and protein-splicing reaction that leads to formation of an EGFP fluorophore. This technique simplified detection of protein interactions, but because of the low splicing efficiency of VDE intein, its sensitivity and screening time were not enough for detecting the protein interactions directly in living cells. In this paper, we have explored the use of the DnaE split intein from Synechocystis sp. PCC6803 for intracellular reconstitution of the split EGFP. We examined efficiency of the fluorophore formation by preparing four different split-EGFP types, among which EGFP dissected at the position between 157 and 158 was found to show the strongest fluorescence intensity upon protein interactions. A time required for the formation of EGFP after protein interactions was only 4 h, as compared to 3 days with the VDE intein. The protein interactions were thereby detected by an in vivo selection and screening assay in Escherichia coli on Luria broth agar plates. This improvement permits versatile designs of screening procedures either for ligands that bind to particular proteins or for molecules or mutations that block particular interactions between two proteins of interest.
Topics: Bacterial Proteins; Cloning, Molecular; DNA Mutational Analysis; Escherichia coli; Green Fluorescent Proteins; Immunoblotting; Luminescent Proteins; Point Mutation; Polymerase Chain Reaction; Protein Splicing; Recombinant Fusion Proteins; Spectrometry, Fluorescence
PubMed: 11791555
DOI: 10.1021/ac010717k -
Biochemical and Biophysical Research... Sep 2009An intein is a polypeptide that interrupts the functional domains of a protein, called the exteins. The intein can facilitate its own excision from the exteins,...
An intein is a polypeptide that interrupts the functional domains of a protein, called the exteins. The intein can facilitate 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 interrupted by three inteins in separate insertion sites. Each intein can facilitate protein splicing when over-expressed in Escherichia coli, with affinity domains serving as the exteins. The influence of the N-terminal flanking residue on the efficiency of splicing is specific to each intein. Each intein has a different downstream nucleophilic residue, and cannot tolerate substitution to a residue of lesser or equal nucleophilicity. The influence of the conserved penultimate His also differs between the inteins.
Topics: Inteins; Protein Splicing; Pyrococcus abyssi; Ribonucleotide Reductases
PubMed: 19577540
DOI: 10.1016/j.bbrc.2009.06.145 -
Trends in Biotechnology May 2005Transgenes in plastids are contained by stringent maternal inheritance in most cultivated plant species and their expression yields high levels of protein with bona fide... (Review)
Review
Transgenes in plastids are contained by stringent maternal inheritance in most cultivated plant species and their expression yields high levels of protein with bona fide structure. Nevertheless, transfer of plastid genes to the nucleus has been reported, with implications for transgene containment. The significance of these transfers will depend on the likelihood that they will become functional nuclear genes. Recently a novel approach, intein-mediated protein trans-splicing, has been demonstrated promising to yield transgenic plants with greatly reduced risk of genetic outcrossing.
Topics: Inteins; Plants, Genetically Modified; Plastids; Protein Splicing; Transgenes
PubMed: 15865996
DOI: 10.1016/j.tibtech.2005.03.006 -
PloS One 2012Inteins catalyze a protein splicing reaction to excise the intein from a precursor protein and join the flanking sequences (exteins) with a peptide bond. In a split...
Inteins catalyze a protein splicing reaction to excise the intein from a precursor protein and join the flanking sequences (exteins) with a peptide bond. In a split intein, the intein fragments (I(N) and I(C)) can reassemble non-covalently to catalyze a trans-splicing reaction that joins the exteins from separate polypeptides. An atypical split intein having a very small I(N) and a large I(C) is particularly useful for joining synthetic peptides with recombinant proteins, which can be a generally useful method of introducing site-specific chemical labeling or modifications into proteins. However, a large I(C) derived from an Ssp DnaX intein was found recently to undergo spontaneous C-cleavage, which raised questions regarding its structure-function and ability to trans-splice. Here, we show that this I(C) could undergo trans-splicing in the presence of I(N), and the trans-splicing activity completely suppressed the C-cleavage activity. We also found that this I(C) could trans-splice with small I(N) sequences derived from two other inteins, showing a cross-reactivity of this atypical split intein. Furthermore, we found that this I(C) could trans-splice even when the I(N) sequence was embedded in a nearly complete intein sequence, suggesting that the small I(N) could project out of the central pocket of the intein to become accessible to the I(C). Overall, these findings uncovered a new atypical split intein that can be valuable for peptide-protein trans-splicing, and they also revealed an interesting structural flexibility and cross-reactivity at the active site of this intein.
Topics: Amino Acid Sequence; Inteins; Mutation; Protein Splicing; Proteins
PubMed: 23024818
DOI: 10.1371/journal.pone.0045355 -
Trends in Biochemical Sciences Jun 1999
Review
Topics: Evolution, Molecular; Models, Genetic; Protein Engineering; Protein Splicing
PubMed: 10366843
DOI: 10.1016/s0968-0004(99)01403-6 -
Journal of Magnetic Resonance (San... May 2022Protein trans-splicing catalyzed by split inteins has been used for segmental isotopic labeling of proteins for alleviating the complexity of NMR signals. Whereas...
Protein trans-splicing catalyzed by split inteins has been used for segmental isotopic labeling of proteins for alleviating the complexity of NMR signals. Whereas inteins spontaneously trigger protein splicing upon protein folding, inteins from extremely halophilic organisms require a high salinity condition to induce protein splicing. We designed and created a salt-inducible intein from the widely used DnaE intein from Nostoc punctiforme by introducing 29 mutations, which required a lower salt concentration than naturally occurring halo-obligate inteins. We determined the NMR solution structure of the engineered salt-inducible DnaE intein in 2 M NaCl, showing the essentially identical three-dimensional structure to the original one, albeit it unfolds without salts. The NMR structure of a halo-obligate intein under high salinity suggests that the stabilization of the active folded conformation is not a mere result of various intramolecular interactions but the subtle energy balance from the complex interactions, including the solvation energy, which involve waters, ions, co-solutes, and protein polypeptide chains.
Topics: DNA Polymerase III; Inteins; Magnetic Resonance Spectroscopy; Nostoc; Protein Splicing
PubMed: 35398651
DOI: 10.1016/j.jmr.2022.107195