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International Journal of Biological... Sep 2021Atypical S1 and S11 split inteins have been used for N-terminal or C-terminal protein labeling. Here we reported a novel site-specific internal protein labeling method...
Atypical S1 and S11 split inteins have been used for N-terminal or C-terminal protein labeling. Here we reported a novel site-specific internal protein labeling method based on two atypical split inteins, Ter DnaE3 S11 and Rma DnaB S1. Protein-peptide trans-splicing activity was first demonstrated in vitro between a short peptide (Flag tag, FLAG) and two recombinant proteins (Maltose binding protein, MBP, and Thioredoxin, Trx) by trans-splicing between MBP-TE3S11N (MBP-N fragment of Ter DnaE3 S11), TE3S11C-FLAG-RBS1N (C fragment of Ter DnaE3 S11-FLAG-N fragment of Rma DnaB S1), and RBS1C-Trx (C fragment of Rma DnaB S1-Trx). To minimize the middle synthetic peptide (TE3S11C-linker-RBS1N), we reduced the number of native extein amino acids, which may play a role in protein trans-splicing. The results showed at least 3 (CKG) native extein amino acids were required for detectable trans-splicing activity. This method was further demonstrated to be effective in facilitating the incorporation of fluorescent probe (FITC) to the internal site of recombinant protein, generating the FITC-labeled protein. Besides the fluorescent group, these two split inteins can also be useful for adding any desirable chemical groups into a protein of interest, which may include biotin, modified and unnatural amino acids, or drug molecules.
Topics: Fluorescein-5-isothiocyanate; Inteins; Maltose-Binding Proteins; Oligopeptides; Protein Engineering; Protein Splicing; Thioredoxins; Trans-Splicing
PubMed: 34246886
DOI: 10.1016/j.ijbiomac.2021.07.009 -
Molekuliarnaia Biologiia 2007Protein splicing is a post-translational autocatalytic excision of internal protein sequence (intein) with the subsequent ligation of the flanking polypeptides... (Review)
Review
Protein splicing is a post-translational autocatalytic excision of internal protein sequence (intein) with the subsequent ligation of the flanking polypeptides (exteins). This process doesn't require any cofactors or enzymes, which distinguish it from other variants of protein processing. Protein splicing is catalyzed by Hint-domain - internal intein's domain. In review the main molecular mechanisms of this process are described. Function of analogous Hint-domains of other proteins families (Hh-proteins, BIL-domains etc) are considered. The analysis of inteins characteristic to different branches of life illustrates the role of horizontal transfer in intein's distribution and evolution. A possible role of inteins in regulation of different cell processes is discussed.
Topics: Amino Acid Sequence; Animals; Catalysis; Endonucleases; Inteins; Protein Splicing; Protein Structure, Tertiary
PubMed: 17514899
DOI: No ID Found -
IUBMB Life Aug 2005Protein splicing is a posttranslational cellular process, in which an intervening protein sequence (intein) is self-catalytically excised out from a nascent protein... (Review)
Review
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 to produce two mature enzymes. This unique reaction was first discovered from studies of the structure and expression of the VMA1 gene in Saccharomyces cerevisiae. VMA1 consists of a single open reading frame and yet comprises two independent genetic information for Vma1p (a catalytic 70-kDa subunit of the vacuolar H+-ATPase) and VDE (a 50-kDa DNA endonuclease) as an in-frame spliced insert in the gene. Subsequent studies have demonstrated that protein splicing is not unique for the VMA1 precursor and there are many operons in nature, which implement genetic information editing at protein level. To elucidate its precise reaction mechanisms from a viewpoint of structure-directed chemistry, a series of crystal structural studies has been carried out with the use of splicing-inactive and slowly spliceable precursors of VMA1 recombinants. One precursor structure revealed that the N-terminal junction of the introduced extein polypeptide forms an intermediate containing a five-membered thiazolidine ring. The other precursor structures showed spliced products with a linkage between the N- and C-extein segments. This article summarizes biochemical and structural studies on a self-catalytic mechanism for protein splicing that is triggered and terminated solely via thiazolidine intermediates with tetrahedral configurations formed within the splicing sites where proton ingress and egress are driven by balanced protonation and deprotonation.
Topics: Amino Acid Sequence; Crystallography; Exteins; Models, Molecular; Molecular Sequence Data; Protein Splicing; Proton-Translocating ATPases; Saccharomyces cerevisiae Proteins; Thiazoles
PubMed: 16118114
DOI: 10.1080/15216540500215499 -
Molecular BioSystems Nov 2010Obtaining insights into the molecular structure and dynamics of a protein by NMR spectroscopy and other in-solution biophysical methods relies heavily on the... (Review)
Review
Obtaining insights into the molecular structure and dynamics of a protein by NMR spectroscopy and other in-solution biophysical methods relies heavily on the incorporation of isotopic labels or other chemical modifications such as fluorescent groups into the protein of interest. These types of modifications can be elegantly achieved with the use of split inteins in a site- and/or region-specific manner. Split inteins are split derivatives of the protein splicing element intein, and catalyze the formation of a peptide bond between two proteins. Recent progress in split intein engineering provided the opportunity to also perform peptide bond formation between a protein and a chemically synthesized peptide. We review the current state-of-the-art in preparing segmental isotope-labeled proteins for NMR spectroscopy, and highlight the importance of split intein orthogonality for the ligation of a protein from multiple fragments. Furthermore, we use split intein-mediated site-specific fluorescent labeling as a framework to illustrate the general usefulness of split inteins for custom protein modifications in the realm of structural biology. We also address some limitations of split intein technology, and offer constructive advice to overcome these shortcomings.
Topics: Amino Acid Sequence; Fluorescent Dyes; Inteins; Isotope Labeling; Molecular Sequence Data; Protein Engineering; Protein Splicing; Trans-Splicing
PubMed: 20820635
DOI: 10.1039/c0mb00034e -
Chemistry & Biology Mar 1997An increasing number of proteins are thought to self-splice post-translationally on the level of the polypeptide, producing two separate proteins from one gene, neither... (Review)
Review
An increasing number of proteins are thought to self-splice post-translationally on the level of the polypeptide, producing two separate proteins from one gene, neither of which is the protein predicted from the gene sequence. The recent elucidation of the mechanism of splicing has led to the identification of a number of post-translational protein modifications that use similar chemical pathways.
Topics: Animals; Humans; Protein Processing, Post-Translational; Protein Splicing; Proteins
PubMed: 9115411
DOI: 10.1016/s1074-5521(97)90287-8 -
Annual Review of Biochemistry 2000Protein splicing is a form of posttranslational processing that consists of the excision of an intervening polypeptide sequence, the intein, from a protein, accompanied... (Comparative Study)
Comparative Study Review
Protein splicing is a form of posttranslational processing that consists of the excision of an intervening polypeptide sequence, the intein, from a protein, accompanied by the concomitant joining of the flanking polypeptide sequences, the exteins, by a peptide bond. It requires neither cofactors nor auxiliary enzymes and involves a series of four intramolecular reactions, the first three of which occur at a single catalytic center of the intein. Protein splicing can be modulated by mutation and converted to highly specific self-cleavage and protein ligation reactions that are useful protein engineering tools. Some of the reactions characteristic of protein splicing also occur in other forms of protein autoprocessing, ranging from peptide bond cleavage to conjugation with nonprotein moieties. These mechanistic similarities may be the result of convergent evolution, but in at least one case-hedgehog protein autoprocessing-there is definitely a close evolutionary relationship to protein splicing.
Topics: Amino Acid Sequence; Asparagine; Bacterial Proteins; Conserved Sequence; Esterification; Fungal Proteins; Gene Expression; Models, Molecular; Mutation; Mycobacterium xenopi; Protein Splicing; Saccharomyces cerevisiae
PubMed: 10966466
DOI: 10.1146/annurev.biochem.69.1.447 -
Advanced Drug Delivery Reviews Sep 2009The judicious application of intein technologies to biological problems has resulted in powerful tools for biomedical research. Inteins are intervening sequences that... (Review)
Review
The judicious application of intein technologies to biological problems has resulted in powerful tools for biomedical research. Inteins are intervening sequences that excise themselves from precursor proteins and ligate the surrounding sequences. Variations of intein chemistry have been used to create tagless protein purification strategies, specifically label expressed proteins for biochemical assays, design biosensors, produce microarrays, and synthesize cyclic peptide libraries for inhibitor studies. Moreover, recent advances in small molecule triggered protein splicing allow for tunable post-translational control of protein function in vivo. Inteins are now positioned as an essential tool to study the mechanism of disease progression and validate drug candidates. Yet these tiny proteins have more tricks to play. Recent progress in gene therapy and drug targeting suggest a bright future where split inteins mediate in vivo reconstruction of large therapeutic proteins and target drugs to a specified site of action. Inteins are rapidly becoming valuable tools for drug discovery and drug delivery.
Topics: Anti-Infective Agents; Biosensing Techniques; Drug Discovery; Genetic Therapy; Inteins; Peptides, Cyclic; Protein Splicing; Proteins
PubMed: 19442693
DOI: 10.1016/j.addr.2009.04.021 -
Biochemistry Feb 2022Protein splicing is a post-translational process by which an intervening protein, or an intein, catalyzes its own excision from flanking polypeptides, or exteins,...
Protein splicing is a post-translational process by which an intervening protein, or an intein, catalyzes its own excision from flanking polypeptides, or exteins, coupled to extein ligation. Four inteins interrupt the MCM helicase of the halophile , two of which are mini-inteins that lack a homing endonuclease. Both inteins can be overexpressed in and purified as unspliced precursors; splicing can be induced by incubation with salt. However, one intein can splice in 0.5 M NaCl , whereas the other splices efficiently only in buffer containing over 2 M NaCl; the organism also requires high salt to grow, with the standard growth media containing over 3 M NaCl and about 0.75 M magnesium salts. Consistent with this difference in salt-dependent activity, an intein-containing precursor protein with both inteins promotes conditional alternative protein splicing (CAPS) to yield different spliced products dependent on the salt concentration. Native Trp fluorescence of the inteins suggests that the difference in activity may be due to partial unfolding of the inteins at lower salt concentrations. This differential salt sensitivity of intein activity may provide a useful mechanism for halophiles to respond to environmental changes.
Topics: Archaeal Proteins; Escherichia coli; Exteins; Halobacteriaceae; Inteins; Minichromosome Maintenance Proteins; Peptides; Protein Precursors; Protein Splicing
PubMed: 35073064
DOI: 10.1021/acs.biochem.1c00788 -
Methods in Enzymology 2021Conventional site-directed mutagenesis and genetic code expansion approaches have been instrumental in providing detailed functional and pharmacological insight into...
Conventional site-directed mutagenesis and genetic code expansion approaches have been instrumental in providing detailed functional and pharmacological insight into membrane proteins such as ion channels. Recently, this has increasingly been complemented by semi-synthetic strategies, in which part of the protein is generated synthetically. This means a vast range of chemical modifications, including non-canonical amino acids (ncAA), backbone modifications, chemical handles, fluorescent or spectroscopic labels and any combination of these can be incorporated. Among these approaches, protein trans-splicing (PTS) is particularly promising for protein reconstitution in live cells. It relies on one or more split inteins, which can spontaneously and covalently link flanking peptide or protein sequences. Here, we describe the use of PTS and its variant tandem PTS (tPTS) in semi-synthesis of ion channels in Xenopus laevis oocytes to incorporate ncAAs, post-translational modifications or metabolically stable mimics thereof. This strategy has the potential to expand the type and number of modifications in ion channel research.
Topics: Inteins; Ion Channels; Peptides; Protein Engineering; Protein Splicing; Trans-Splicing
PubMed: 34120713
DOI: 10.1016/bs.mie.2021.01.028 -
Microbiology Spectrum Oct 2021tervening pro, or inteins, are mobile genetic elements that are translated within host polypeptides and removed at the protein level by splicing. In protein splicing, a...
tervening pro, or inteins, are mobile genetic elements that are translated within host polypeptides and removed at the protein level by splicing. In protein splicing, a self-mediated reaction removes the intein, leaving a peptide bond in place. While protein splicing can proceed in the absence of external cofactors, several examples of conditional protein splicing (CPS) have emerged. In CPS, the rate and accuracy of splicing are highly dependent on environmental conditions. Because the activity of the intein-containing host protein is compromised prior to splicing and inteins are highly abundant in the microbial world, CPS represents an emerging form of posttranslational regulation that is potentially widespread in microbes. Reactive chlorine species (RCS) are highly potent oxidants encountered by bacteria in a variety of natural environments, including within cells of the mammalian innate immune system. Here, we demonstrate that two naturally occurring RCS, namely, hypochlorous acid (the active compound in bleach) and -chlorotaurine, can reversibly block splicing of DnaB inteins from Mycobacterium leprae and Mycobacterium smegmatis . Further, using a reporter that monitors DnaB intein activity within M. smegmatis, we show that DnaB protein splicing is inhibited by RCS in the native host. DnaB, an essential replicative helicase, is the most common intein-housing protein in bacteria. These results add to the growing list of environmental conditions that are relevant to the survival of the intein-containing host and influence protein splicing, as well as suggesting a novel mycobacterial response to RCS. We propose a model in which DnaB splicing, and therefore replication, is paused when these mycobacteria encounter RCS. Inteins are both widespread and abundant in microbes, including within several bacterial and fungal pathogens. Inteins are domains translated within host proteins and removed at the protein level by splicing. Traditionally considered molecular parasites, some inteins have emerged in recent years as adaptive posttranslational regulatory elements. Several studies have demonstrated CPS, in which the rate and accuracy of protein splicing, and thus host protein functions, are responsive to environmental conditions relevant to the intein-containing organism. In this work, we demonstrate that two naturally occurring RCS, including the active compound in household bleach, reversibly inhibit protein splicing of Mycobacterium leprae and Mycobacterium smegmatis DnaB inteins. In addition to describing a new physiologically relevant condition that can temporarily inhibit protein splicing, this study suggests a novel stress response in Mycobacterium, a bacterial genus of tremendous importance to humans.
Topics: Chloramines; Chlorine; DNA Replication; DnaB Helicases; Gene Expression Regulation, Bacterial; Hypochlorous Acid; Inteins; Mycobacterium leprae; Mycobacterium smegmatis; Oxidants; Oxidation-Reduction; Protein Splicing; Reactive Oxygen Species; Taurine
PubMed: 34549994
DOI: 10.1128/Spectrum.00301-21