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Chembiochem : a European Journal of... Aug 2019Protein-splicing domains are frequently used engineering tools that find application in the in vivo and in vitro ligation of protein domains. Directed evolution is among...
Protein-splicing domains are frequently used engineering tools that find application in the in vivo and in vitro ligation of protein domains. Directed evolution is among the most promising technologies used to advance this technology. However, the available screening systems for protein-splicing activity are associated with bottlenecks such as the selection of pseudo-positive clones arising from off-pathway reaction products or fragment complementation. Herein, we report a stringent screening method for protein-splicing activity in cis and trans, that exclusively selects productively splicing domains. By fusing splicing domains to an intrinsically disordered region of the antidote from the Escherichia coli CcdA/CcdB type II toxin/antitoxin system, we linked protein splicing to cell survival. The screen allows selecting novel cis- and trans-splicing inteins catalyzing productive highly efficient protein splicing, for example, from directed-evolution approaches or the natural intein sequence space.
Topics: Antitoxins; Bacterial Proteins; Cell Survival; Drug Evaluation, Preclinical; Escherichia coli; Models, Molecular; Protein Splicing
PubMed: 30963690
DOI: 10.1002/cbic.201900139 -
Annual Review of Plant Biology 2005Protein splicing elements, termed inteins, have been discovered in all the domains of life. Basic research on inteins has led to a greater understanding of how they... (Review)
Review
Protein splicing elements, termed inteins, have been discovered in all the domains of life. Basic research on inteins has led to a greater understanding of how they mediate the protein splicing process. Because inteins are natural protein engineering elements they have been harnessed for use in a number of applications, including protein purification, protein semisynthesis, and in vivo and in vitro protein modifications. This review focuses on the use of inteins in plants. A split-gene technique utilizes inteins to reconstitute the activity of a transgene product with the goal of limiting the spread of transgenes from a genetically modified plant to a weedy relative. Furthermore, merging the intein tag for protein purification with the large protein yields possible with plants has the potential to produce pharmaceutically important proteins. Finally, relevant techniques that may be used in plants in the future are discussed.
Topics: Acetolactate Synthase; Plant Proteins; Plants; Protein Splicing; Transgenes
PubMed: 15862101
DOI: 10.1146/annurev.arplant.56.032604.144242 -
MBio Jun 2024tervening pro (inteins) are translated as subdomains within host proteins and removed through an intein-driven splicing reaction where the flanking sequences (exteins)...
tervening pro (inteins) are translated as subdomains within host proteins and removed through an intein-driven splicing reaction where the flanking sequences (exteins) are joined with a peptide bond. Previously, we developed a self-removing translation reporter for labeling Ebola virus (EBOV). In this reporter, an intein (RadA) containing the fluorescent protein ZsGreen (ZsG) is inserted within the EBOV protein VP30. Upon VP30-RadA-ZsG expression from the viral genome, RadA-ZsG is removed from VP30 through the protein splicing activity of RadA, generating functional, non-tagged VP30 and functional ZsGreen. While incorporation of our VP30-RadA-ZsG fusion reporter into recombinant EBOV (rEBOV-RadA-ZsG) resulted in an infectious virus that expresses ZsG upon infection of cells, this virus displayed a replication defect compared to wild-type EBOV, which might be the result of insufficient RadA splicing. Here, we demonstrate that the serial passaging of rEBOV-RadA-ZsG in human cells led to an increase in replication efficiency compared to unpassaged rEBOV-RadA-ZsG. Sequencing of passaged viruses revealed intein-specific mutations. These mutations improve intein activity in both prokaryotic and eukaryotic systems, as well as in multiple extein contexts. Taken together, our findings offer a novel means to select for inteins with enhanced catalytic properties that appear independent of extein context and expression system.IMPORTANCEtervening pro (inteins) are self-removing protein elements that have been utilized to develop a variety of innovative protein engineering technologies. Here, we report the isolation of inteins with improved catalytic activity through viral passaging. Specifically, we inserted a highly active intein within an essential protein of Ebola virus and serially passaged this recombinant virus, which led to intein-specific hyper-activity mutations. The identified mutations showed improved intein activity within both bacterial and eukaryotic expression systems and in multiple extein contexts. These results present a new strategy for developing inteins with improved splicing activity.
Topics: Humans; Inteins; Protein Splicing; Ebolavirus; Virus Replication; Viral Proteins; Genes, Reporter
PubMed: 38780266
DOI: 10.1128/mbio.00984-24 -
The Journal of Biological Chemistry May 2014Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave themselves from larger precursor proteins and ligate their flanking... (Review)
Review
Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave themselves from larger precursor proteins and ligate their flanking polypeptides (exteins) through a multistep chemical reaction. First thought to be an anomaly found in only a few organisms, protein splicing by inteins has since been observed in microorganisms from all domains of life. Despite this broad phylogenetic distribution, all inteins share common structural features such as a horseshoe-like pseudo two-fold symmetric fold, several canonical sequence motifs, and similar splicing mechanisms. Intriguingly, the splicing efficiencies and substrate specificity of different inteins vary considerably, reflecting subtle changes in the chemical mechanism of splicing, linked to their local structure and dynamics. As intein chemistry has widespread use in protein chemistry, understanding the structural and dynamical aspects of inteins is crucial for intein engineering and the improvement of intein-based technologies.
Topics: Amino Acids; Catalytic Domain; Inteins; Models, Molecular; Mutation; Protein Folding; Protein Precursors; Protein Splicing; Protein Structure, Tertiary; Proteins
PubMed: 24695731
DOI: 10.1074/jbc.R113.540302 -
Current Opinion in Biotechnology Aug 2000Protein splicing elements, termed inteins, provide a fertile source for innovative biotechnology tools. First harnessed for protein purification, inteins are now used to... (Review)
Review
Protein splicing elements, termed inteins, provide a fertile source for innovative biotechnology tools. First harnessed for protein purification, inteins are now used to express cytotoxic proteins, to segmentally modify or label proteins, to cyclize proteins or peptides, to study structure-activity relationships and to generate reactive polypeptide termini in expressed proteins for an expanding list of chemoselective reactions, including protein ligation.
Topics: Biotechnology; Protein Splicing; Recombinant Proteins
PubMed: 10975457
DOI: 10.1016/s0958-1669(00)00113-0 -
Proceedings of the Japan Academy.... 2009Twenty years ago, evidence that one gene produces two enzymes via protein splicing emerged from structural and expression studies of the VMA1 gene in Saccharomyces... (Review)
Review
Twenty years ago, evidence that one gene produces two enzymes via protein splicing emerged from structural and expression studies of the VMA1 gene in Saccharomyces cerevisiae. VMA1 consists of a single open reading frame and contains 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. Protein splicing is a posttranslational cellular process, in which an intervening polypeptide termed as the VMA1 intein is self-catalytically excised out from a nascent 120-kDa VMA1 precursor and two flanking polypeptides of the N- and C-exteins are ligated to produce the mature Vma1p. Subsequent studies have demonstrated that protein splicing is not unique to the VMA1 precursor and there are many operons in nature, which implement genetic information editing at protein level. To elucidate its structure-directed chemical mechanisms, a series of biochemical and crystal structural studies has been carried out with the use of various VMA1 recombinants. This article summarizes a VDE-mediated 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: Exteins; Inteins; Molecular Sequence Data; Peptides; Protein Splicing; Proton-Translocating ATPases; Saccharomyces cerevisiae Proteins; Thiazolidines
PubMed: 19907126
DOI: 10.2183/pjab.85.409 -
Ukrains'kyi Biokhimichnyi Zhurnal (1999... 2005Inteins are internal polypeptide sequences that are posttranslationally excised from a protein precursor by a self-catalyzed protein-splicing reaction. Most of inteins... (Review)
Review
Inteins are internal polypeptide sequences that are posttranslationally excised from a protein precursor by a self-catalyzed protein-splicing reaction. Most of inteins consist of N- and C-terminal protein splicing domain and central endonuclease domain. The endonuclease domain can initiate mobility of the intein gene, this process being named intein homing. This review is focused on the recent data about the structure and function of inteins. Main intein-mediated protein-engineering applications, such as protein purification, ligation and cyclization, new forms of biosensors, are presented.
Topics: Inteins; Models, Molecular; Protein Splicing; Proteins
PubMed: 16568601
DOI: No ID Found -
Genetic Engineering 2001
Review
Topics: Amino Acid Motifs; Evolution, Molecular; Molecular Biology; Protein Splicing
PubMed: 11570103
DOI: 10.1007/0-306-47572-3_10 -
MBio Jan 2018Inteins are intervening proteins that undergo an autocatalytic splicing reaction that ligates flanking host protein sequences termed exteins. Some intein-containing...
Inteins are intervening proteins that undergo an autocatalytic splicing reaction that ligates flanking host protein sequences termed exteins. Some intein-containing proteins have evolved to couple splicing to environmental signals; this represents a new form of posttranslational regulation. Of particular interest is RadA from the archaeon , for which long-range intein-extein interactions block splicing, requiring temperature and single-stranded DNA (ssDNA) substrate to splice rapidly and accurately. Here, we report that splicing of the intein-containing RadA from another archaeon, , is activated by significantly lower temperatures than is RadA, consistent with differences in their growth environments. Investigation into variations between and RadA inteins led to the discovery that a nonconserved region (NCR) of the intein, a flexible loop where a homing endonuclease previously resided, is critical to splicing. Deletion of the NCR leads to a substantial loss in the rate and accuracy of RadA splicing only within native exteins. The influence of the NCR deletion can be largely overcome by ssDNA, demonstrating that the splicing-competent conformation can be achieved. We present a model whereby the NCR is a flexible hinge which acts as a switch by controlling distant intein-extein interactions that inhibit active site assembly. These results speak to the repurposing of the vestigial endonuclease loop to control an intein-extein partnership, which ultimately allows exquisite adaptation of protein splicing upon changes in the environment. Inteins are mobile genetic elements that interrupt coding sequences (exteins) and are removed by protein splicing. They are abundant elements in microbes, and recent work has demonstrated that protein splicing can be controlled by environmental cues, including the substrate of the intein-containing protein. Here, we describe an intein-extein collaboration that controls temperature-induced splicing of RadA from two archaea and how variation in this intein-extein partnership results in fine-tuning of splicing to closely match the environment. Specifically, we found that a small sequence difference between the two inteins, a flexible loop that likely once housed a homing endonuclease used for intein mobility, acts as a switch to control intein-extein interactions that block splicing. Our results argue strongly that some inteins have evolved away from a purely parasitic lifestyle to control the activity of host proteins, representing a new form of posttranslational regulation that is potentially widespread in the microbial world.
Topics: Archaeal Proteins; DNA-Binding Proteins; Exteins; Inteins; Models, Biological; Models, Molecular; Protein Splicing; Pyrococcus horikoshii; Sequence Deletion; Temperature; Thermococcus
PubMed: 29382734
DOI: 10.1128/mBio.02304-17 -
Journal of Molecular Biology Dec 2023Optogenetics has emerged as a powerful tool for spatiotemporal control of biological processes. Near-infrared (NIR) light, with its low phototoxicity and deep tissue...
Optogenetics has emerged as a powerful tool for spatiotemporal control of biological processes. Near-infrared (NIR) light, with its low phototoxicity and deep tissue penetration, holds particular promise. However, the optogenetic control of polypeptide bond formation has not yet been developed. In this study, we introduce a NIR optogenetic module for conditional protein splicing (CPS) based on the gp41-1 intein. We optimized the module to minimize background signals in the darkness and to maximize the contrast between light and dark conditions. Next, we engineered a NIR CPS gene expression system based on the protein ligation of a transcription factor. We applied the NIR CPS for light-triggered protein cleavage to activate gasdermin D, a pore-forming protein that induces pyroptotic cell death. Our NIR CPS optogenetic module represents a promising tool for controlling molecular processes through covalent protein linkage and cleavage.
Topics: Protein Splicing; Optogenetics; Inteins; Gene Expression Regulation
PubMed: 37949312
DOI: 10.1016/j.jmb.2023.168360