1,503 results on '"Inteins"'
Search Results
2. Split Gp41‐1 intein splicing as a model to evaluate the cellular location of the oncosuppressor Maspin in an in vitro model of osteosarcoma.
- Author
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Mariano, Alessia, Di Cristofano, Samuele, Raimondo, Domenico, and Scotto d'Abusco, Anna
- Subjects
- *
TUMOR suppressor proteins , *FLUORESCENT proteins , *CHIMERIC proteins , *PEPTIDES , *OSTEOSARCOMA , *GENETIC code - Abstract
Inteins are proteins involved in the protein splicing mechanism, an autoprocessing event, where sequences (exteins) separated by inteins become ligated each other after recombination. Two kinds of inteins have been described, contiguous inteins and split inteins. The former ones are transcribed and translated as a single peptide along with their exteins, while the latter are fragmented between two different genes and are transcribed and translated separately. The aim of this study is to establish a method to obtain a fluorescent eukaryotic protein to analyze its cellular localization, using the natural split gp41‐1 inteins. We chose natural split inteins due to their distribution in all three domains of life. Two constructs were prepared, one containing the N‐terminal split intein along with the N‐moiety of the Red Fluorescent Protein (RFP) and a second construct containing the C‐terminal of split intein, the C‐moiety of RFP and the gene coding for Maspin, a tumor suppressor protein. The trans‐splicing was verified by transfecting both N‐terminal and C‐terminal constructs into mammalian cells. The success of the recombination event was highlighted through the fluorescence produced by reconstituted RFP after recombination, along with the overlap of the red fluorescence produced by recombined RFP and the green fluorescence produced by the hybridization of the recombinant Maspin with a specific antibody. In conclusion, we opted to use this mechanism of recombination to obtain a fluorescent Maspin instead to express a large fusion protein, considering that it could interfere with Maspin's structure and function. Significance statement: This manuscript focuses on the use of Ultra‐Fast Split Gp41‐1 intein to obtain a fluorescent eukaryotic protein, Maspin, a tumor suppressor protein, and to analyze its cellular localization. This method is based on protein recombination, using split intein and Red Fluorescent Protein. The protein recombination produces a fluorescent Maspin to be localized into cell compartments. The advantage of this method to obtain a fluorescent protein instead to express a large fusion protein is the ability to avoiding the interference that a large protein could have on Maspin structure and function. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
3. Optimization strategies and advances in the research and development of AAV‐based gene therapy to deliver large transgenes.
- Author
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Kolesnik, Valeria V., Nurtdinov, Ruslan F., Oloruntimehin, Ezekiel Sola, Karabelsky, Alexander V., and Malogolovkin, Alexander S.
- Subjects
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GENE therapy , *GENETIC transformation , *TRANSGENES , *GENE expression , *PROTEIN engineering , *RESEARCH & development - Abstract
Adeno‐associated virus (AAV)‐based therapies are recognized as one of the most potent next‐generation treatments for inherited and genetic diseases. However, several biological and technological aspects of AAV vectors remain a critical issue for their widespread clinical application. Among them, the limited capacity of the AAV genome significantly hinders the development of AAV‐based gene therapy. In this context, genetically modified transgenes compatible with AAV are opening up new opportunities for unlimited gene therapies for many genetic disorders. Recent advances in de novo protein design and remodelling are paving the way for new, more efficient and targeted gene therapeutics. Using computational and genetic tools, AAV expression cassette and transgenic DNA can be split, miniaturized, shuffled or created from scratch to mediate efficient gene transfer into targeted cells. In this review, we highlight recent advances in AAV‐based gene therapy with a focus on its use in translational research. We summarize recent research and development in gene therapy, with an emphasis on large transgenes (>4.8 kb) and optimizing strategies applied by biomedical companies in the research pipeline. We critically discuss the prospects for AAV‐based treatment and some emerging challenges. We anticipate that the continued development of novel computational tools will lead to rapid advances in basic gene therapy research and translational studies. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
4. Recombinant Production of TP4-LYC1, A New Chimeric Peptide with Targeted Cytotoxicity to HeLa Cells.
- Author
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Pour, Hanieh Mohammad, Jahanian-Najafabadi, Ali, and Shafiee, Fatemeh
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ANTI-inflammatory agents , *BIOTECHNOLOGY , *CELL physiology , *APOPTOSIS , *ARTHROPOD venom , *CELL lines , *PEPTIDES , *RECOMBINANT proteins , *ENDOTHELIAL cells , *AMINO acids , *CELL survival , *PHARMACODYNAMICS - Abstract
Background: Tilapia Piscidin 4 (TP4) showed potential anti-tumor effects against various cancer cells. Lycosine-1 (LYC1), is another Antimicrobial Peptides (AMP) from spider venom with targeted penetration to cancer cells without any adverse effects on normal cells. The aim of this study was to produce a soluble recombinant fusion peptide in order to diminish the cytotoxicity of TP4 against normal cells. Methods: In order to express of TP4-LYC-1, TP4, and LYC1 in fusion to the inteins1/2 of pTWIN-1 vector, induction condition was optimized to earn soluble peptides. Autocleavage induction of inteins1/2 was performed based on IMPACT® manual and their effect on cell viability of HeLa and HUVEC cells was surveyed by MTT assay. Results: The best condition for accessing the most soluble peptide in fusion to the inteins was approximately similar for all three peptides (0.1 mM of IPTG, at 22°C). After the induction of self-cleavage of inteins, a band in 3, 3, and 6 kDa was observed on tricine- SDS-PAGE. The IC50 values of TP4-LYC1 and TP4 against HeLa cells were calculated as 0.83, and 2.75 µM, respectively. Conclusion: In the present study, a novel chimeric peptide, TP4-LYC1, was successfully produced. This fusion protein can act as a safe bio-molecule with potent cytotoxic effects against cancer cells, but the penetration ability and determination of cell death mechanism must be performed in order to have more precise view on the apoptosis induction of this recombinant peptide. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
5. Optimization strategies and advances in the research and development of AAV‐based gene therapy to deliver large transgenes
- Author
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Valeria V. Kolesnik, Ruslan F. Nurtdinov, Ezekiel Sola Oloruntimehin, Alexander V. Karabelsky, and Alexander S. Malogolovkin
- Subjects
exons remodelling ,gene editing ,gene therapies ,inteins ,minigenes ,protein design ,Medicine (General) ,R5-920 - Abstract
Abstract Adeno‐associated virus (AAV)‐based therapies are recognized as one of the most potent next‐generation treatments for inherited and genetic diseases. However, several biological and technological aspects of AAV vectors remain a critical issue for their widespread clinical application. Among them, the limited capacity of the AAV genome significantly hinders the development of AAV‐based gene therapy. In this context, genetically modified transgenes compatible with AAV are opening up new opportunities for unlimited gene therapies for many genetic disorders. Recent advances in de novo protein design and remodelling are paving the way for new, more efficient and targeted gene therapeutics. Using computational and genetic tools, AAV expression cassette and transgenic DNA can be split, miniaturized, shuffled or created from scratch to mediate efficient gene transfer into targeted cells. In this review, we highlight recent advances in AAV‐based gene therapy with a focus on its use in translational research. We summarize recent research and development in gene therapy, with an emphasis on large transgenes (>4.8 kb) and optimizing strategies applied by biomedical companies in the research pipeline. We critically discuss the prospects for AAV‐based treatment and some emerging challenges. We anticipate that the continued development of novel computational tools will lead to rapid advances in basic gene therapy research and translational studies.
- Published
- 2024
- Full Text
- View/download PDF
6. The Evolutionary History of a DNA Methylase Reveals Frequent Horizontal Transfer and Within-Gene Recombination.
- Author
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Gosselin, Sophia P., Arsenault, Danielle R., Jennings, Catherine A., and Gogarten, Johann Peter
- Subjects
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INTRONS , *HORIZONTAL gene transfer , *MOBILE genetic elements , *METHYLTRANSFERASES , *DNA modification & restriction , *DNA , *DNA insertion elements - Abstract
Inteins, often referred to as protein introns, are highly mobile genetic elements that invade conserved genes throughout the tree of life. Inteins have been found to invade a wide variety of key genes within actinophages. While in the process of conducting a survey of these inteins in actinophages, we discovered that one protein family of methylases contained a putative intein, and two other unique insertion elements. These methylases are known to occur commonly in phages as orphan methylases (possibly as a form of resistance to restriction–modification systems). We found that the methylase family is not conserved within phage clusters and has a disparate distribution across divergent phage groups. We determined that two of the three insertion elements have a patchy distribution within the methylase protein family. Additionally, we found that the third insertion element is likely a second homing endonuclease, and that all three elements (the intein, the homing endonuclease, and what we refer to as the ShiLan domain) have different insertion sites that are conserved in the methylase gene family. Furthermore, we find strong evidence that both the intein and ShiLan domain are partaking in long-distance horizontal gene transfer events between divergent methylases in disparate phage hosts within the already dispersed methylase distribution. The reticulate evolutionary history of methylases and their insertion elements reveals high rates of gene transfer and within-gene recombination in actinophages. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
7. Inteins: A Swiss army knife for synthetic biology.
- Author
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Anastassov, Stanislav, Filo, Maurice, and Khammash, Mustafa
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POST-translational modification , *SYNTHETIC biology , *LOGIC circuits , *VIRUS diseases , *CANCER treatment , *MEDICAL screening - Abstract
Inteins are proteins found in nature that execute protein splicing. Among them, split inteins stand out for their versatility and adaptability, presenting creative solutions for addressing intricate challenges in various biological applications. Their exquisite attributes, including compactness, reliability, orthogonality, low toxicity, and irreversibility, make them of interest to various fields including synthetic biology, biotechnology and biomedicine. In this review, we delve into the inherent challenges of using inteins, present approaches for overcoming these challenges, and detail their reliable use for specific cellular tasks. We will discuss the use of conditional inteins in areas like cancer therapy, drug screening, patterning, infection treatment, diagnostics and biocontainment. Additionally, we will underscore the potential of inteins in executing basic logical operations with practical implications. We conclude by showcasing their potential in crafting complex genetic circuits for performing computations and feedback control that achieves robust perfect adaptation. • Inteins enable protein post-translational modifications without external cofactors. • Inteins are utilized for bio-computations including logic gates and feedback control. • Inteins are used for drug screening, targeted protein delivery and biocontainment. • They have shown promise in diagnostics, cancer therapy and treating viral infections. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
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8. Inteins: Localized Distribution, Gene Regulation, and Protein Engineering for Biological Applications
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Pavankumar, Theetha L
- Subjects
Biological Sciences ,Bioinformatics and Computational Biology ,inteins ,splicing ,Hint domains ,DNA replication ,repair and recombination ,protein engineering ,Microbiology ,Medical microbiology - Abstract
Inteins are self-splicing polypeptides with an ability to excise themselves from flanking host protein regions with remarkable precision; in the process, they ligate flanked host protein fragments. Inteins are distributed sporadically across all three domains of life (bacteria, archaea, and unicellular eukaryotes). However, their apparent localized distribution in DNA replication, repair, and recombination proteins (the 3Rs), particularly in bacteria and archaea, is enigmatic. Our understanding of the localized distribution of inteins in the 3Rs, and their possible regulatory role in such distribution, is still only partial. Nevertheless, understanding the chemistry of post-translational self-splicing of inteins has opened up opportunities for protein chemists to modify, manipulate, and bioengineer proteins. Protein-splicing technology is adapted to a wide range of applications, starting with untagged protein purification, site-specific protein labeling, protein biotinylation, isotope incorporation, peptide cyclization, as an antimicrobial target, and so on. This review is focused on the chemistry of splicing; the localized distribution of inteins, particularly in the 3Rs and their possible role in regulating host protein function; and finally, the use of protein-splicing technology in various protein engineering applications.
- Published
- 2018
9. Split intein-mediated backbone cyclization enhances the stability and activity of staphylokinase, a potent fibrin-selective plasminogen activator.
- Author
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Baharian A, Ishida H, Sillner C, and Vogel HJ
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- Cyclization, Enzyme Stability, Proteolysis, Plasminogen Activators chemistry, Plasminogen Activators metabolism, Plasminogen Activators pharmacology, Escherichia coli drug effects, Staphylococcus aureus drug effects, Humans, Plasminogen metabolism, Plasminogen chemistry, Fibrinolytic Agents pharmacology, Fibrinolytic Agents chemistry, Inteins, Metalloendopeptidases chemistry, Metalloendopeptidases metabolism, Fibrin chemistry, Fibrin metabolism
- Abstract
Staphylokinase (Sak), a small 15 kDa globular protein that is secreted by certain strains of Staphylococcus aureus, shows a potent fibrin-selective thrombolytic activity. Earlier work has shown that Sak could potentially become a low-cost alternative to currently used thrombolytic agents, such as tissue plasminogen activator (tPA). In attempts to improve its potential for clinical applications, numerous modifications of Sak have already been investigated. Here, we have characterized a novel Sak modification, cyclized Sak (cyc-Sak), which was prepared through split-intein mediated protein backbone cyclization. We have characterized the structure, stability and the activity of cyc-Sak using biophysical techniques, limited proteolysis studies and plasminogen (PG)-activation assays. Our results show that cyc-Sak possesses an identical structure, enhanced stability, resistance to proteolysis by exoproteases and improved PG-activation properties compared to its linear counterpart. It can be over-expressed with high yield in the cytoplasm of Escherichia coli and is easily purified in a two-step process. The intein-mediated cyclization occurs spontaneously in vivo during protein expression and does not necessitate further modification steps after purification of the protein. Furthermore, covalent Sak cyclization could be readily combined with other Sak modifications previously proposed, to generate an effective thrombolytic agent with lower immunogenicity and improved stability and activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2024
- Full Text
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10. Site-directed display of zearalenone lactonase on spilt-intein functionalized nanocarrier for green and efficient detoxification of zearalenone.
- Author
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Zhou C, He N, Lin X, Liu H, Lu Z, and Zhang G
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- Inteins, Zearalenone chemistry
- Abstract
In this study, we prepared a functional organic-inorganic hybrid nanoflower (InHNF) via split intein moiety in a biomineralization process without using organic solvents. InHNF could specifically bind the target enzymes from crude cell lysates within seconds and site-directedly display them on the surface by forming a peptide bond with enzyme's terminal amino acid residue. This unique feature enabled InHNF to increase the specific activity of zearalenone detoxifying enzyme ZHD518 by 40 ∼ 60% at all tested temperatures and prevented enzyme denaturation even under extreme pH conditions (pH 3-11). Furthermore, it exhibited excellent operational stability, with a residual activity of over 70% after eight reaction cycles. Strikingly, InHNF-ZHD518 achieved above 50% ZEN degradation despite the near inactivation of free ZHD518 in beer sample. Overall, InHNF nanocarriers can achieve environmentally friendly, purification-free, and site-directed immobilization of food enzymes and enhance their catalytic properties, making them suitable for a wide range of industrial applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)
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- 2024
- Full Text
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11. Towards targeted Cas9 (CRISPR-Cas) delivery: Preparation of IgG antibody-Cas9 conjugates using a split intein.
- Author
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Pasch T, Bäumer N, Bäumer S, Buchholz F, and Mootz HD
- Subjects
- Humans, CRISPR-Associated Protein 9 genetics, CRISPR-Associated Protein 9 metabolism, CRISPR-Associated Protein 9 chemistry, Inteins, Immunoglobulin G chemistry, Immunoglobulin G genetics, CRISPR-Cas Systems
- Abstract
The CRISPR-Cas9 system has revolutionized the field of genetic engineering, but targeted cellular delivery remains a central problem. The delivery of the preformed ribonuclease-protein (RNP) complex has the advantages of fewer side effects and avoidance of potential permanent effects. We reasoned that an internalizing IgG antibody as a targeting device could address the delivery of Cas9-RNP. We opted for protein trans-splicing mediated by a split intein to facilitate posttranslational conjugation of the two large protein entities. We recently described the cysteine-less CL split intein that efficiently performs under oxidizing conditions and does not interfere with disulfide bonds or thiol bioconjugation chemistries. Using the CL split intein, we report for the first time the ligation of monoclonal IgG antibody precursors, expressed in mammalian cells, and a Cas9 precursor, obtained from bacterial expression. A purified IgG-Cas9 conjugate was loaded with sgRNA to form the active RNP complex and introduced a double-strand break in its target DNA in vitro. Furthermore, a synthetic peptide variant of the short N-terminal split intein precursor proved useful for chemical modification of Cas9. The split intein ligation procedure reported here for IgG-Cas9 provides the first step towards a novel CRISPR-Cas9 targeting approach involving the preformed RNP complex., (© 2024 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)
- Published
- 2024
- Full Text
- View/download PDF
12. Promising properties of cytochrome P450 BM3 reconstituted from separate domains by split intein.
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Yoo SK, Cheong DE, Yoo HS, Choi HJ, Nguyen NA, Yun CH, and Kim GJ
- Subjects
- Protein Domains, Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins metabolism, Inteins, Cytochrome P-450 Enzyme System chemistry, Cytochrome P-450 Enzyme System metabolism, Cytochrome P-450 Enzyme System genetics, Heme chemistry, Heme metabolism, NADPH-Ferrihemoprotein Reductase chemistry, NADPH-Ferrihemoprotein Reductase genetics, NADPH-Ferrihemoprotein Reductase metabolism
- Abstract
Recombinant cytochrome P450 monooxygenases possess significant potential as biocatalysts, and efforts to improve heme content, electron coupling efficiency, and catalytic activity and stability are ongoing. Domain swapping between heme and reductase domains, whether natural or engineered, has thus received increasing attention. Here, we successfully achieved split intein-mediated reconstitution (IMR) of the heme and reductase domains of P450 BM3 both in vitro and in vivo. Intriguingly, the reconstituted enzymes displayed promising properties for practical use. IMR BM3 exhibited a higher heme content (>50 %) and a greater tendency for oligomerization compared to the wild-type enzyme. Moreover, these reconstituted enzymes exhibited a distinct increase in activity ranging from 165 % to 430 % even under the same heme concentrations. The reproducibility of our results strongly suggests that the proposed reconstitution approach could pave a new path for enhancing the catalytic efficiency of related enzymes., Competing Interests: Declaration of competing interest Geun-Joong Kim, Chul-Ho Yun, Dae-Eun Cheong, Su-Kyoung Yoo, and Hye-Ji Choi declare that they have conflict of interest because “recombinant vector and method for producing reconstituted cytochrome P450 oxygenase-reductase fusion protein using the same” has been registered as patents (Korean Patent No. 10-2252056, United States of America Patent No. 11,236,309). Ho-Seok Yoo and Ngoc Anh Nguyen declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)
- Published
- 2024
- Full Text
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13. Ligation of multiple protein domains using orthogonal inteins with non-native splice junctions.
- Author
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Romero-Casañas A, García-Lizarribar A, Castro J, Vilanova M, Benito A, and Ribó M
- Subjects
- Protein Engineering methods, Escherichia coli genetics, Escherichia coli metabolism, Inteins, Protein Splicing, Protein Domains
- Abstract
Protein splicing is a self-catalyzed process in which an internal protein domain (the intein) is excised from its flanking sequences, linking them together with a canonical peptide bond. Trans-inteins are separated in two different precursor polypeptide chains that must assemble to catalytically self-excise and ligate the corresponding flanking exteins to join even when expressed separately either in vitro or in vivo. They are very interesting to construct full proteins from separate domains because their common small size favors chemical synthesis approaches. Therefore, trans-inteins have multiple applications such as protein modification and purification, structural characterization of protein domains or production of intein-based biosensors, among others. For many of these applications, when using more than one trans-intein, orthogonality between them is a critical issue to ensure the proper ligation of the exteins. Here, we confirm the orthogonality (lack of cross-reactivity) of four different trans- or split inteins, gp41-1, gp41-8, IMPDH-1 and NrdJ-1 both in vivo and in vitro, and built different constructs that allow for the sequential fusion of up to four protein fragments into one final spliced product. We have characterized the splicing efficiency of these constructs. All harbor non-native extein residues at the splice junction between the trans-intein and the neighboring exteins, except for the essential Ser + 1. Our results show that it is possible to ligate four different protein domains using inteins gp41-1, IMPDH-1 and NrdJ-1 with non-native extein residues to obtain a final four-domain spliced product with a not negligible yield that keeps its native sequence., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)
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- 2024
- Full Text
- View/download PDF
14. Editorial: Genetically mobile elements repurposed by nature and biotechnologists
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Christopher W. Lennon, Brian P. Callahan, Benoit Cousineau, David R. Edgell, and Marlene Belfort
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mobile genetic elements ,introns ,inteins ,CRISPR ,biotechnology ,Biology (General) ,QH301-705.5 - Published
- 2022
- Full Text
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15. The fingerprint of antimitochondrial antibodies and the etiology of primary biliary cholangitis
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Shuai, Zongwen, Wang, Jinjun, Badamagunta, Madhu, Choi, Jinjung, Yang, Guoxiang, Zhang, Weici, Kenny, Thomas P, Guggenheim, Kathryn, Kurth, Mark J, Ansari, Aftab A, Voss, John, Coppel, Ross L, Invernizzi, Pietro, Leung, Patrick SC, and Gershwin, M Eric
- Subjects
Biomedical and Clinical Sciences ,Clinical Sciences ,Immunology ,Rare Diseases ,Autoimmune Disease ,Antibody Affinity ,Autoantibodies ,Case-Control Studies ,Cholangitis ,Electron Spin Resonance Spectroscopy ,Enzyme-Linked Immunosorbent Assay ,Humans ,Inteins ,Mitochondria ,Pyruvate Dehydrogenase (Lipoamide) ,Xenobiotics ,Medical Biochemistry and Metabolomics ,Gastroenterology & Hepatology ,Clinical sciences - Abstract
The identification of environmental factors that lead to loss of tolerance has been coined the holy grail of autoimmunity. Our work has focused on the reactivity of antimitochondrial autoantibodies (AMA) to chemical xenobiotics and has hypothesized that a modified peptide within PDC-E2, the major mitochondrial autoantigen, will have been immunologically recognized at the time of loss of tolerance. Herein, we successfully applied intein technology to construct a PDC-E2 protein fragment containing amino acid residues 177-314 of PDC-E2 by joining a recombinant peptide spanning residues 177-252 (PDC-228) with a 62-residue synthetic peptide from 253 to 314 (PP), which encompasses PDC-E2 inner lipoyl domain (ILD). We named this intein-constructed fragment PPL. Importantly, PPL, as well as lipoic acid conjugated PPL (LA-PPL) and xenobiotic 2-octynoic acid conjugated PPL (2OA-PPL), are recognized by AMA. Of great importance, AMA has specificity for the 2OA-modified PDC-E2 ILD peptide backbone distinct from antibodies that react with native lipoylated PDC-E2 peptide. Interestingly, this unique AMA subfraction is of the immunoglobulin M isotype and more dominant in early-stage primary biliary cholangitis (PBC), suggesting that exposure to 2OA-PPL-like compounds occurs early in the generation of AMA. To understand the structural basis of this differential recognition, we analyzed PPL, LA-PPL, and 2OA-PPL using electron paramagnetic resonance spectroscopy, with confirmations by enzyme-linked immunosorbent assay, immunoblotting, and affinity antibody analysis. We demonstrate that the conformation of PDC-E2 ILD is altered when conjugated with 2OA, compared to conjugation with lipoic acid.ConclusionA molecular understanding of the conformation of xenobiotic-modified PDC-E2 is critical for understanding xenobiotic modification and loss of tolerance in PBC with widespread implications for a role of environmental chemicals in the induction of autoimmunity. (Hepatology 2017;65:1670-1682).
- Published
- 2017
16. The Evolution of Intein-Based Affinity Methods as Reflected in 30 years of Patent History
- Author
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Sai Vivek Prabhala, Izabela Gierach, and David W. Wood
- Subjects
inteins ,protein purification ,patents ,split inteins ,protein engineering ,Biology (General) ,QH301-705.5 - Abstract
Self-cleaving affinity tags, based on engineered intein protein domains, have been touted as a universal single step purification platform for tagless non-mAb proteins. These approaches provide all of the power and flexibility of tag-based affinity methods, but deliver a tagless target protein suitable for clinical applications without complex process development. This combination of features might accelerate and de-risk biopharmaceutical development by bridging early discovery to full-scale manufacturing under a single platform. Despite this profound promise, intein-based technologies have yet to reach their full potential. This review examines the evolution of intein-based purification methods in the light of several significant intein patents filed over the last 3 decades. Illustrated with actual key figures from each of the relevant patents, key advances are described with a focus on applications in basic research and biopharmaceutical production. Suggestions for extending intein-based purification systems to emerging therapies and non-protein applications are presented as concluding remarks.
- Published
- 2022
- Full Text
- View/download PDF
17. The Evolutionary History of a DNA Methylase Reveals Frequent Horizontal Transfer and Within-Gene Recombination
- Author
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Sophia P. Gosselin, Danielle R. Arsenault, Catherine A. Jennings, and Johann Peter Gogarten
- Subjects
actinophage ,actinobacteriophage ,inteins ,LAGLIDADG endonuclease ,homing ,horizontal gene transfer ,Genetics ,QH426-470 - Abstract
Inteins, often referred to as protein introns, are highly mobile genetic elements that invade conserved genes throughout the tree of life. Inteins have been found to invade a wide variety of key genes within actinophages. While in the process of conducting a survey of these inteins in actinophages, we discovered that one protein family of methylases contained a putative intein, and two other unique insertion elements. These methylases are known to occur commonly in phages as orphan methylases (possibly as a form of resistance to restriction–modification systems). We found that the methylase family is not conserved within phage clusters and has a disparate distribution across divergent phage groups. We determined that two of the three insertion elements have a patchy distribution within the methylase protein family. Additionally, we found that the third insertion element is likely a second homing endonuclease, and that all three elements (the intein, the homing endonuclease, and what we refer to as the ShiLan domain) have different insertion sites that are conserved in the methylase gene family. Furthermore, we find strong evidence that both the intein and ShiLan domain are partaking in long-distance horizontal gene transfer events between divergent methylases in disparate phage hosts within the already dispersed methylase distribution. The reticulate evolutionary history of methylases and their insertion elements reveals high rates of gene transfer and within-gene recombination in actinophages.
- Published
- 2023
- Full Text
- View/download PDF
18. Protein Splicing of Inteins: A Powerful Tool in Synthetic Biology
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Hao Wang, Lin Wang, Baihua Zhong, and Zhuojun Dai
- Subjects
inteins ,synthetic biology ,living therapeutics ,protein engineering ,split inteins ,Biotechnology ,TP248.13-248.65 - Abstract
Inteins are protein segments that are capable of enabling the ligation of flanking extein into a new protein, a process known as protein splicing. Since its discovery, inteins have become powerful biotechnological tools for applications such as protein engineering. In the last 10 years, the development in synthetic biology has further endowed inteins with enhanced functions and diverse utilizations. Here we review these efforts and discuss the future directions.
- Published
- 2022
- Full Text
- View/download PDF
19. One-Step Purification and N-Terminal Functionalization of Bioactive Proteins via Atypically Split Inteins.
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Gharios R, Li A, Kopyeva I, Francis RM, and DeForest CA
- Subjects
- Green Fluorescent Proteins chemistry, Green Fluorescent Proteins metabolism, Green Fluorescent Proteins genetics, beta-Lactamases metabolism, beta-Lactamases chemistry, Luminescent Proteins chemistry, Epidermal Growth Factor metabolism, Epidermal Growth Factor chemistry, Red Fluorescent Protein, Proteins chemistry, Inteins
- Abstract
Site-specific installation of non-natural functionality onto proteins has enabled countless applications in biotechnology, chemical biology, and biomaterials science. Though the N-terminus is an attractive derivatization location, prior methodologies targeting this site have suffered from low selectivity, a limited selection of potential chemical modifications, and/or challenges associated with divergent protein purification/modification steps. In this work, we harness the atypically split VidaL intein to simultaneously N-functionalize and purify homogeneous protein populations in a single step. Our method─referred to as VidaL-tagged expression and protein ligation (VEPL)─enables modular and scalable production of N-terminally modified proteins with native bioactivity. Demonstrating its flexibility and ease of use, we employ VEPL to combinatorially install 4 distinct (multi)functional handles (e.g., biotin, alkyne, fluorophores) to the N-terminus of 4 proteins that span three different classes: fluorescent (Enhanced Green Fluorescent Protein, mCherry), enzymatic (β-lactamase), and growth factor (epidermal growth factor). Moving forward, we anticipate that VEPL's ability to rapidly generate and isolate N-modified proteins will prove useful across the growing fields of applied chemical biology.
- Published
- 2024
- Full Text
- View/download PDF
20. Production of native recombinant proteins using a novel split intein affinity technology.
- Author
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Clifford R, Lindman S, Zhu J, Luo E, Delmar J, Tao Y, Ren K, Lara A, Cayatte C, McTamney P, O'Connor E, and Öhman J
- Subjects
- Humans, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Spike Glycoprotein, Coronavirus metabolism, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus genetics, Spike Glycoprotein, Coronavirus isolation & purification, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Recombinant Fusion Proteins isolation & purification, SARS-CoV-2 genetics, SARS-CoV-2 chemistry, Interleukin-1beta metabolism, Interleukin-1beta genetics, Inteins, Chromatography, Affinity methods
- Abstract
Affinity tags are frequently engineered into recombinant proteins to facilitate purification. Although this technique is powerful, removal of the tag is desired because the tag can interfere with biological activity and can potentially increase the immunogenicity of therapeutic proteins. Tag removal is complex, as it requires adding expensive protease enzymes. To overcome this limitation, split intein based affinity purification systems have been developed in which a C
C -intein tag is engineered into a protein of interest for binding to a NC -intein peptide ligand fixed to a chromatographic support. Tag removal in these systems is achieved by creating an active intein-complex during protein capture, which triggers a precise self-cleavage reaction. In this work, we show applications of a new split intein system, Cytiva™ ProteinSelect™. One advantage of the new system is that the NC -intein ligand can be robustly produced and conjugated to large volumes of resin for production of gram scale proteins. SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager in this work were successfully captured on the affinity resin and scaled 10-fold. Another advantage of this system is the ability to sanitize the resin with sodium hydroxide without loosing the 10-20 g/L binding capacity. Binding studies with IL-1b and IFNAR-1 ECD showed that the resin can be regenerated and sanitized for up to 50 cycles without loosing binding capacity. Additionally, after several cycles of sanitization, binding capacity was retained for the SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager. As with other split intein systems, optimization was needed to achieve ideal expression and recovery. The N-terminal amino acid sequence of the protein of interest required engineering to enable the cleavage reaction. Additionally, ensuring the stability of the CC -intein tag was important to prevent premature cleavage or truncation. Controlling the hold time of the expression product and the prevention of protease activity prior to purification was needed. These results demonstrate the feasibility of the Cytiva™ ProteinSelect™ system to be used in academic and industrial research and development laboratories for the purification of novel proteins expressed in either bacterial or mammalian systems., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Johan Ohman has patent #WO2021099607A1 licensed to CYTIVA BIOPROCESS R&D AB. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)- Published
- 2024
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21. Int&in: A machine learning-based web server for active split site identification in inteins.
- Author
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Schmitz M, Ballestin JB, Liang J, Tomas F, Freist L, Voigt K, Di Ventura B, and Öztürk MA
- Subjects
- Catalytic Domain, Inteins, Machine Learning, Protein Splicing, Internet, Software
- Abstract
Inteins are proteins that excise themselves out of host proteins and ligate the flanking polypeptides in an auto-catalytic process called protein splicing. In nature, inteins are either contiguous or split. In the case of split inteins, the two fragments must first form a complex for the splicing to occur. Contiguous inteins have previously been artificially split in two fragments because split inteins allow for distinct applications than contiguous ones. Even naturally split inteins have been split at unnatural split sites to obtain fragments with reduced affinity for one another, which are useful to create conditional inteins or to study protein-protein interactions. So far, split sites in inteins have been heuristically identified. We developed Int&in, a web server freely available for academic research (https://intein.biologie.uni-freiburg.de) that runs a machine learning model using logistic regression to predict active and inactive split sites in inteins with high accuracy. The model was trained on a dataset of 126 split sites generated using the gp41-1, Npu DnaE and CL inteins and validated using 97 split sites extracted from the literature. Despite the limited data size, the model, which uses various protein structural features, as well as sequence conservation information, achieves an accuracy of 0.79 and 0.78 for the training and testing sets, respectively. We envision Int&in will facilitate the engineering of novel split inteins for applications in synthetic and cell biology., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)
- Published
- 2024
- Full Text
- View/download PDF
22. Split Proteins and Reassembly Modules for Biological Applications.
- Author
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Bae J, Kim J, Choi J, Lee H, and Koh M
- Subjects
- Humans, Inteins, Proteins metabolism, Proteins chemistry, Protein Engineering, Biosensing Techniques
- Abstract
Split systems, modular entities enabling controlled biological processes, have become instrumental in biological research. This review highlights their utility across applications like gene regulation, protein interaction identification, and biosensor development. Covering significant progress over the last decade, it revisits traditional split proteins such as GFP, luciferase, and inteins, and explores advancements in technologies like Cas proteins and base editors. We also examine reassembly modules and their applications in diverse fields, from gene regulation to therapeutic innovation. This review offers a comprehensive perspective on the recent evolution of split systems in biological research., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)
- Published
- 2024
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23. Metal effect on intein splicing: A review.
- Author
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Panda, Sunita, Nanda, Ananya, Nasker, Sourya Subhra, Sen, Debjani, Mehra, Ashwaria, and Nayak, Sasmita
- Subjects
- *
PROTEIN precursors , *METALS , *METAL compounds , *DRUG design - Abstract
Inteins are intervening polypeptides that interrupt the functional domains of several important proteins across the three domains of life. Inteins excise themselves from the precursor protein, ligating concomitant extein residues in a process called protein splicing. Post-translational auto-removal of inteins remain critical for the generation of active proteins. The perspective of inteins in science is a robust field of research, however fundamental studies centralized upon splicing regulatory mechanism are imperative for addressing more intricate issues. Controlled engineering of intein splicing has many applications; intein inhibition can facilitate novel drug design, while activation of intein splicing is exploited in protein purification. This paper provides a comprehensive review of the past and recent advances in the splicing regulation via metal-intein interaction. We compare the behavior of different metal ions on diverse intein systems. Though metals such as Zn, Cu, Pt, Cd, Co, Ni exhibit intein inhibitory effect heterogeneously on different inteins, divalent metal ions such as Ca and Mg fail to do so. The observed diversity in the metal-intein interaction arises mostly due to intein polymorphism and variations in atomic structure of metals. A mechanistic understanding of intein regulation by metals in native as well as synthetically engineered intein systems may yield potent intein inhibitors via direct or indirect approach. [Display omitted] • Inteins interrupt the functional domains of essential proteins in microorganisms. • Intein splicing is critical for generation of active proteins in microorganisms. • Conserved catalytic and non-catalytic residues mediate metal-intein interaction. • Metal ions and compounds exhibit heterogenous effect on diverse intein systems. • Intein inhibition by metals may provide a novel approach for drug development. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
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24. Gene Therapy Blueprints for NeuroAIDS
- Author
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Rodriguez, Hector E., Lakshmi, Seetha, Somboonwit, Charurut, Oxner, Asa, Guerra, Lucy, Addisu, Anteneh, Gutierrez, Louise, Sinnott, John T., Nilofer, Christina, Kangueane, Pandjassarame, Shapshak, Paul, Shapshak, Paul, editor, Levine, Andrew J., editor, Foley, Brian T., editor, Somboonwit, Charurut, editor, Singer, Elyse, editor, Chiappelli, Francesco, editor, and Sinnott, John T., editor
- Published
- 2017
- Full Text
- View/download PDF
25. Navigating the Cellular Crowd : Physicochemical Properties of Protein Surfaces as Evolved Interaction Guides
- Abstract
The cellular interior is characterised by high concentrations of macromolecules. Compared with dilute conditions, the crowd modifies proteins' ability to fold, diffuse and, ultimately, carry out their biological functions. Cellular fitness depends on ensuring an adequate balance between interactivity and diffusivity. In this thesis, I discuss how a colloidal description of the cell highlights the central role of electrostatics in protein surface optimisation. By recognising that the modulation of protein-protein interactions concerns the whole proteome, I map the physicochemical preferences of cellular organisms across taxonomic and ecological divisions. Moreover, I propose that all surface residues participate in tuning protein interactions to the correct affinity, within a continuum that spans several orders of magnitude. Finally, I turn to horizontally spreading inteins to gauge the strength of the selective pressures acting on protein surfaces.
- Published
- 2023
26. Navigating the Cellular Crowd : Physicochemical Properties of Protein Surfaces as Evolved Interaction Guides
- Abstract
The cellular interior is characterised by high concentrations of macromolecules. Compared with dilute conditions, the crowd modifies proteins' ability to fold, diffuse and, ultimately, carry out their biological functions. Cellular fitness depends on ensuring an adequate balance between interactivity and diffusivity. In this thesis, I discuss how a colloidal description of the cell highlights the central role of electrostatics in protein surface optimisation. By recognising that the modulation of protein-protein interactions concerns the whole proteome, I map the physicochemical preferences of cellular organisms across taxonomic and ecological divisions. Moreover, I propose that all surface residues participate in tuning protein interactions to the correct affinity, within a continuum that spans several orders of magnitude. Finally, I turn to horizontally spreading inteins to gauge the strength of the selective pressures acting on protein surfaces.
- Published
- 2023
27. Navigating the Cellular Crowd : Physicochemical Properties of Protein Surfaces as Evolved Interaction Guides
- Abstract
The cellular interior is characterised by high concentrations of macromolecules. Compared with dilute conditions, the crowd modifies proteins' ability to fold, diffuse and, ultimately, carry out their biological functions. Cellular fitness depends on ensuring an adequate balance between interactivity and diffusivity. In this thesis, I discuss how a colloidal description of the cell highlights the central role of electrostatics in protein surface optimisation. By recognising that the modulation of protein-protein interactions concerns the whole proteome, I map the physicochemical preferences of cellular organisms across taxonomic and ecological divisions. Moreover, I propose that all surface residues participate in tuning protein interactions to the correct affinity, within a continuum that spans several orders of magnitude. Finally, I turn to horizontally spreading inteins to gauge the strength of the selective pressures acting on protein surfaces.
- Published
- 2023
28. Navigating the Cellular Crowd : Physicochemical Properties of Protein Surfaces as Evolved Interaction Guides
- Abstract
The cellular interior is characterised by high concentrations of macromolecules. Compared with dilute conditions, the crowd modifies proteins' ability to fold, diffuse and, ultimately, carry out their biological functions. Cellular fitness depends on ensuring an adequate balance between interactivity and diffusivity. In this thesis, I discuss how a colloidal description of the cell highlights the central role of electrostatics in protein surface optimisation. By recognising that the modulation of protein-protein interactions concerns the whole proteome, I map the physicochemical preferences of cellular organisms across taxonomic and ecological divisions. Moreover, I propose that all surface residues participate in tuning protein interactions to the correct affinity, within a continuum that spans several orders of magnitude. Finally, I turn to horizontally spreading inteins to gauge the strength of the selective pressures acting on protein surfaces.
- Published
- 2023
29. Efficient expression and purification of tag-free recombinant human procalcitonin (hPCT) with precise sequence in E.coli.
- Author
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Nakagawa M, Tomioka Y, and Akuta T
- Subjects
- Humans, Recombinant Fusion Proteins genetics, Recombinant Proteins chemistry, Inteins, Chromatography, Affinity methods, Escherichia coli genetics, Escherichia coli metabolism, Procalcitonin metabolism
- Abstract
We present an efficient method for expression and purification of recombinant human procalcitonin (hPCT) in E. coli T7 express LysY/Iq cells, ensuring precise N- and C-terminal amino acid sequences. Our method involves fusing codon-optimized cDNA with two distinct tag sequences: eXact tag and chitin binding domain (CBD) tag. To purify the protein, we employ a two-step affinity chromatography process. Firstly, we utilize the N-terminal Profinity eXact tag and purify the protein through Profinity eXact-affinity column chromatography using a resin on which a mutant subtilisin protease was immobilized. The eXact tag was removed by adding NaF to activate the enzyme. Subsequently, the digested sample containing C-terminal CBD tag is directly loaded for the second step of chitin affinity chromatography. Elution is achieved through dithiothreitol (DTT)-catalyzed self-cleavage of the intein sequence from the fusion protein. As a result, the target protein is selectively recovered in the flow-through, completely tag-free, with a purity exceeding 95%. To ensure high purity and eliminate potential contaminants, we effectively remove E. coli host DNA and endotoxins through a combination of streptomycin sulfate, Triton X-114, and ammonium sulfate treatment. The exceptional level of purity obtained eliminates the need for further purification steps in most applications. This highly purified hPCT can be used as a calibrator in procalcitonin or calcitonin immunoassays. Notably, our approach effectively manages small peptides that are prone to degradation by E. coli host proteases, offering a robust solution for various research and application requirements., (Copyright © 2023 Elsevier Inc. All rights reserved.)
- Published
- 2024
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- View/download PDF
30. A Chemical Counterpart to the Resolution Step of Nature's Intein-Mediated Protein Splicing.
- Author
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Dhayalan B, Kent SBH, and Fetter-Pruneda I
- Subjects
- Proteins, Peptides chemistry, RNA Splicing, Inteins, Protein Splicing
- Abstract
In the course of an attempted total chemical synthesis of the ant insulin-like peptide-2 (ILP2) protein molecule, specific cleavage of a backbone peptide bond in a branched ester-linked polypeptide chain with concomitant peptide splicing was observed. The side reaction was investigated in model compounds. Here, we postulate a chemical mechanism for this novel polypeptide backbone cleavage reaction as a chemical counterpart to the resolution step of biochemical intein-mediated protein splicing.
- Published
- 2024
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31. Recombinant Production of TP4-LYC1, A New Chimeric Peptide with Targeted Cytotoxicity to HeLa Cells.
- Author
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Mohammad Pour H, Jahanian-Najafabadi A, and Shafiee F
- Abstract
Background: Tilapia Piscidin 4 (TP4) showed potential anti-tumor effects against various cancer cells. Lycosine-1 (LYC1), is another Antimicrobial Peptides (AMP) from spider venom with targeted penetration to cancer cells without any adverse effects on normal cells. The aim of this study was to produce a soluble recombinant fusion peptide in order to diminish the cytotoxicity of TP4 against normal cells., Methods: In order to express of TP4-LYC-1, TP4, and LYC1 in fusion to the inteins1/2 of pTWIN-1 vector, induction condition was optimized to earn soluble peptides. Auto-cleavage induction of inteins1/2 was performed based on IMPACT
® manual and their effect on cell viability of HeLa and HUVEC cells was surveyed by MTT assay., Results: The best condition for accessing the most soluble peptide in fusion to the inteins was approximately similar for all three peptides (0.1 mM of IPTG, at 22 °C ). After the induction of self-cleavage of inteins, a band in 3, 3, and 6 kDa was observed on tricine-SDS-PAGE. The IC50 values of TP4-LYC1 and TP4 against HeLa cells were calculated as 0.83, and 2.75 μM , respectively., Conclusion: In the present study, a novel chimeric peptide, TP4-LYC1, was successfully produced. This fusion protein can act as a safe bio-molecule with potent cytotoxic effects against cancer cells, but the penetration ability and determination of cell death mechanism must be performed in order to have more precise view on the apoptosis induction of this recombinant peptide., (Copyright© 2024 Avicenna Research Institute.)- Published
- 2024
- Full Text
- View/download PDF
32. Feasibility of Domain Segmentation of B19V VP1u Using Intein Technology for Structural Studies.
- Author
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Lakshmanan RV, Agbandje-McKenna M, and McKenna R
- Subjects
- Protein Domains, Parvovirus B19, Human genetics, Parvovirus B19, Human chemistry, Nostoc genetics, Nostoc enzymology, Nostoc chemistry, Phospholipases A2 chemistry, Phospholipases A2 genetics, Phospholipases A2 metabolism, Circular Dichroism, Humans, Inteins genetics, Capsid Proteins chemistry, Capsid Proteins genetics, Capsid Proteins metabolism, Escherichia coli genetics, Escherichia coli metabolism
- Abstract
Introduction: Parvovirus B19 (B19V) is a human pathogen, and the minor capsid protein of B19V possesses a unique N terminus called VP1u that plays a crucial role in the life cycle of the virus., Objectives: The objective of this study was to develop a method for domain segmentation of B19 VP1u using intein technology, particularly its receptor binding domain (RBD) and phospholipase A2 (PLA
2 ) domain., Methods: RBD and PLA2 domains of VP1u were each fused to the DnaE split inteins derived from the Nostoc punctiforme . Each of these precursor proteins was expressed in E. coli . Combining the purified precursors in equal molar ratios resulted in the formation of full-length VP1u. Furthermore, Circular Dichroism (CD) spectroscopy and PLA2 assays were used to probe the structure and activity of the newly formed protein., Results: The CD spectrum of the full length VP1u confirmed the secondary structure of protein, while the PLA2 assay indicated minimal disruption in enzymatic activity., Conclusion: This method would allow for the selective incorporation of NMR-active isotopes into either of the VP1u domains, which can reduce signal overlap in NMR structural determination studies., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)- Published
- 2024
- Full Text
- View/download PDF
33. Calcimycin Inhibits
- Author
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Anil Mathew, Tharappel, Zhong, Li, Yan Chun, Zhu, Xiangmeng, Wu, Sudha, Chaturvedi, Qing-Yu, Zhang, and Hongmin, Li
- Subjects
Fungal Proteins ,Mice ,Antifungal Agents ,Cryptococcus neoformans ,Animals ,Humans ,Cryptococcosis ,Sequence Alignment ,Calcimycin ,Inteins - Abstract
Drug resistance is a significant concern in the treatment of diseases, including cryptococcosis caused by
- Published
- 2023
34. Mobile DNAs and switching mating types in yeast
- Author
-
Laura N Rusche
- Subjects
homing endonucleases ,inteins ,mating-type switching ,Torulaspora ,Lachancea ,yeast ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The gene that allows budding yeast cells to switch their mating type evolved from a newly discovered family of genes named weird HO.
- Published
- 2020
- Full Text
- View/download PDF
35. The yeast mating-type switching endonuclease HO is a domesticated member of an unorthodox homing genetic element family
- Author
-
Aisling Y Coughlan, Lisa Lombardi, Stephanie Braun-Galleani, Alexandre AR Martos, Virginie Galeote, Frédéric Bigey, Sylvie Dequin, Kevin P Byrne, and Kenneth H Wolfe
- Subjects
homing endonucleases ,inteins ,mating-type switching ,torulaspora ,lachancea ,yeast ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The mating-type switching endonuclease HO plays a central role in the natural life cycle of Saccharomyces cerevisiae, but its evolutionary origin is unknown. HO is a recent addition to yeast genomes, present in only a few genera close to Saccharomyces. Here we show that HO is structurally and phylogenetically related to a family of unorthodox homing genetic elements found in Torulaspora and Lachancea yeasts. These WHO elements home into the aldolase gene FBA1, replacing its 3' end each time they integrate. They resemble inteins but they operate by a different mechanism that does not require protein splicing. We show that a WHO protein cleaves Torulaspora delbrueckii FBA1 efficiently and in an allele-specific manner, leading to DNA repair by gene conversion or NHEJ. The DNA rearrangement steps during WHO element homing are very similar to those during mating-type switching, and indicate that HO is a domesticated WHO-like element.
- Published
- 2020
- Full Text
- View/download PDF
36. NMR Structure and Dynamics of TonB Investigated by Scar-Less Segmental Isotopic Labeling Using a Salt-Inducible Split Intein
- Author
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Annika Ciragan, Sofia M. Backlund, Kornelia M. Mikula, Hannes M. Beyer, O. H. Samuli Ollila, and Hideo Iwaï
- Subjects
inteins ,protein ligation ,NMR spectroscopy ,segmental isotopic labeling ,TonB ,intrinsically disordered protein ,Chemistry ,QD1-999 - Abstract
The growing understanding of partially unfolded proteins increasingly points to their biological relevance in allosteric regulation, complex formation, and protein design. However, the structural characterization of disordered proteins remains challenging. NMR methods can access both the dynamics and structures of such proteins, yet suffering from a high degeneracy of NMR signals. Here, we overcame this bottleneck utilizing a salt-inducible split intein to produce segmentally isotope-labeled samples with the native sequence, including the ligation junction. With this technique, we investigated the NMR structure and conformational dynamics of TonB from Helicobacter pylori in the presence of a proline-rich low complexity region. Spin relaxation experiments suggest that the several nano-second time scale dynamics of the C-terminal domain (CTD) is almost independent of the faster pico-to-nanosecond dynamics of the low complexity central region (LCCR). Our results demonstrate the utility of segmental isotopic labeling for proteins with heterogenous dynamics such as TonB and could advance NMR studies of other partially unfolded proteins.
- Published
- 2020
- Full Text
- View/download PDF
37. Cellular Synthesis and X‐ray Crystal Structure of a Designed Protein Heterocatenane.
- Author
-
Liu, Yajie, Duan, Zelin, Fang, Jing, Zhang, Fan, Xiao, Junyu, and Zhang, Wen‐Bin
- Subjects
- *
PROTEIN structure , *CRYSTAL structure , *GEL permeation chromatography , *X-rays , *PROTEIN synthesis , *LIQUID chromatography-mass spectrometry - Abstract
Herein, we report the biosynthesis of protein heterocatenanes using a programmed sequence of multiple post‐translational processing events including intramolecular chain entanglement, in situ backbone cleavage, and spontaneous cyclization. The approach is general, autonomous, and can obviate the need for any additional enzymes. The catenane topology was convincingly proven using a combination of SDS‐PAGE, LC‐MS, size exclusion chromatography, controlled proteolytic digestion, and protein crystallography. The X‐ray crystal structure clearly shows two mechanically interlocked protein rings with intact folded domains. It opens new avenues in the nascent field of protein‐topology engineering. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
38. Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System.
- Author
-
De La Cruz Pech-Canul, Angel, Carrillo-Campos, Javier, de Lourdes Ballinas-Casarrubias, María, Solis-Oviedo, Rosa Lidia, Hernández-Rascón, Selena Karina, Hernández-Ochoa, León Raúl, Gutiérrez-Méndez, Néstor, and García-Triana, Antonio
- Subjects
- *
MANGANESE peroxidase , *PHANEROCHAETE chrysosporium , *PROTEIN expression , *CHIMERIC proteins , *AFFINITY chromatography , *MANGANESE porphyrins , *PEROXIDASE - Abstract
Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn2+ to Mn3+, a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli-expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn2+ [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
39. Crystal structures of CDC21-1 inteins from hyperthermophilic archaea reveal the selection mechanism for the highly conserved homing endonuclease insertion site.
- Author
-
Beyer, Hannes M., Mikula, Kornelia M., Kudling, Tatiana V., and Iwaï, Hideo
- Subjects
- *
ENDONUCLEASES , *CRYSTAL structure , *MOBILE genetic elements , *ARCHAEBACTERIA , *CELL division , *HORIZONTAL gene transfer - Abstract
Self-splicing inteins are mobile genetic elements invading host genes via nested homing endonuclease (HEN) domains. All HEN domains residing within inteins are inserted at a highly conserved insertion site. A purifying selection mechanism directing the location of the HEN insertion site has not yet been identified. In this work, we solved the three-dimensional crystal structures of two inteins inserted in the cell division control protein 21 of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii. A comparison between the structures provides the structural basis for the thermo-stabilization mechanism of inteins that have lost the HEN domain during evolution. The presence of an entire extein domain in the intein structure from Pyrococcus horikoshii suggests the selection mechanism for the highly conserved HEN insertion point. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
40. Split intein-mediated selection of cells containing two plasmids using a single antibiotic.
- Author
-
Palanisamy, Navaneethan, Degen, Anna, Morath, Anna, Ballestin Ballestin, Jara, Juraske, Claudia, Öztürk, Mehmet Ali, Sprenger, Georg A., Youn, Jung-Won, Schamel, Wolfgang W., and Di Ventura, Barbara
- Subjects
INTEINS ,CELLS ,PLASMIDS ,ANTIBIOTICS ,BACTERIAL cells - Abstract
To build or dissect complex pathways in bacteria and mammalian cells, it is often necessary to recur to at least two plasmids, for instance harboring orthogonal inducible promoters. Here we present SiMPl, a method based on rationally designed split enzymes and intein-mediated protein trans-splicing, allowing the selection of cells carrying two plasmids with a single antibiotic. We show that, compared to the traditional method based on two antibiotics, SiMPl increases the production of the antimicrobial non-ribosomal peptide indigoidine and the non-proteinogenic aromatic amino acid para-amino-L-phenylalanine from bacteria. Using a human T cell line, we employ SiMPl to obtain a highly pure population of cells double positive for the two chains of the T cell receptor, TCRα and TCRβ, using a single antibiotic. SiMPl has profound implications for metabolic engineering and for constructing complex synthetic circuits in bacteria and mammalian cells. Engineering cell lines often requires multiple plasmids with different selection markers. Here the authors present SiMPl, a method based on rationally engineered split enzymes which get reconstituted via intein-mediated protein splicing to maintain two plasmids using a single antibiotic. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
41. Fused Split Inteins: Tools for Introducing Multiple Protein Modifications
- Author
-
Lim, Byung Joon, Berkeley, Raymond F, and Debelouchina, Galia T
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Biotechnology ,Generic health relevance ,Amino Acids ,Bacterial Proteins ,Chromatography ,High Pressure Liquid ,Cloning ,Molecular ,Codon ,Terminator ,Cyanobacteria ,DNA Polymerase III ,Disulfides ,Escherichia coli ,Gene Expression ,Genetic Vectors ,Hydrolysis ,Inteins ,Lysine ,Norbornanes ,Protein Engineering ,Protein Folding ,Recombinant Fusion Proteins ,Spectrometry ,Mass ,Electrospray Ionization ,Ubiquitin ,Intein ,Amber suppression ,Unnatural amino acid incorporation ,Protein modification ,Tagless protein purification ,Other Chemical Sciences ,Developmental Biology ,Biochemistry and cell biology ,Medicinal and biomolecular chemistry - Abstract
The split inteins from the DnaE cyanobacterial family are efficient and versatile tools for protein engineering and chemical biology applications. Their ultrafast splicing kinetics allow for the efficient production of native proteins from two separate polypeptides both in vitro and in cells. They can also be used to generate proteins with C-terminal thioesters for downstream applications. In this chapter, we describe a method based on a genetically fused version of the DnaE intein Npu for the preparation of doubly modified proteins through recombinant expression. In particular, we provide protocols for the recombinant production of modified ubiquitin through amber suppression where fused Npu is used (1) as a traceless purification tag or (2) as a protein engineering tool to introduce C-terminal modifications for subsequent attachment to other proteins of interest. Our purification protocol allows for quick and facile separation of truncated products and eliminates the need for engineering protease cleavage sites. Our approach can be easily adapted to different proteins and applications where the simultaneous presence of internal and C-terminal modifications is desirable.
- Published
- 2020
42. An Intein-Mediated Split–nCas9 System for Base Editing in Plants
- Author
-
Guoliang Yuan, Haiwei Lu, Kuntal De, Md Mahmudul Hassan, Yang Liu, Yi Li, Wellington Muchero, Paul E. Abraham, Gerald A. Tuskan, and Xiaohan Yang
- Subjects
Gene Editing ,Biomedical Engineering ,General Medicine ,CRISPR-Cas Systems ,Plants ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Genome, Plant ,Inteins - Abstract
Virus-assisted delivery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system represents a promising approach for editing plant genomes. Among the CRISPR/Cas systems, CRISPR/Cas9 is most widely used; however, to pack the relatively large size of the CRISPR/Cas9 system into viral vectors with confined packaging capacity is challenging. To address this technical challenge, we developed a strategy based on split inteins that splits the required CRISPR/Cas9 components across a dual-vector system. The CRISPR/Cas reassembles into an active form following co-infection to achieve targeted genome editing in plant cells. An intein-mediated split system was adapted and optimized in plant cells by a successful demonstration of split-eYGFPuv expression. Using a plant-based biosensor, we demonstrated for the first time that the split-nCas9 can induce efficient base editing in plant cells. We identified several split sites for future biodesign strategies. Overall, this strategy provides new opportunities to bridge different CRISPR/Cas9 tools including base editor, prime editor, and CRISPR activation with virus-mediated gene editing.
- Published
- 2022
43. Massive intein content in Anaeramoeba reveals aspects of intein mobility in eukaryotes.
- Author
-
Gallot-Lavallée L, Jerlström-Hultqvist J, Zegarra-Vidarte P, Salas-Leiva DE, Stairs CW, Čepička I, Roger AJ, and Archibald JM
- Subjects
- Eukaryota genetics, Proteins genetics, Genome, Inteins genetics, Protein Splicing
- Abstract
Inteins are self-splicing protein elements found in viruses and all three domains of life. How the DNA encoding these selfish elements spreads within and between genomes is poorly understood, particularly in eukaryotes where inteins are scarce. Here, we show that the nuclear genomes of three strains of Anaeramoeba encode between 45 and 103 inteins, in stark contrast to four found in the most intein-rich eukaryotic genome described previously. The Anaeramoeba inteins reside in a wide range of proteins, only some of which correspond to intein-containing proteins in other eukaryotes, prokaryotes, and viruses. Our data also suggest that viruses have contributed to the spread of inteins in Anaeramoeba and the colonization of new alleles. The persistence of Anaeramoeba inteins might be partly explained by intragenomic movement of intein-encoding regions from gene to gene. Our intein dataset greatly expands the spectrum of intein-containing proteins and provides insights into the evolution of inteins in eukaryotes., Competing Interests: Competing interests statement:The authors declare no competing interest.
- Published
- 2023
- Full Text
- View/download PDF
44. A novel protein purification scheme based on salt inducible self-assembling peptides.
- Author
-
Zeng G, Zheng Y, Xiang Y, Liu R, Yang X, and Lin Z
- Subjects
- Humans, Proteins metabolism, Inteins, Protein Processing, Post-Translational, Sodium Chloride metabolism, Escherichia coli metabolism, Peptides metabolism
- Abstract
Background: Protein purification remains a critical need for biosciences and biotechnology. It frequently requires multiple rounds of chromatographic steps that are expensive and time-consuming. Our lab previously reported a cleavable self-aggregating tag (cSAT) scheme for streamlined protein expression and purification. The tag consists of a self-assembling peptide (SAP) and a controllable self-cleaving intein. The SAP drives the target protein into an active aggregate, then by intein-mediated cleavage, the target protein is released. Here we report a novel cSAT scheme in which the self-assembling peptide is replaced with a salt inducible self-assembling peptide. This allows a target protein to be expressed first in the soluble form, and the addition of salt then drives the target protein into the aggregated form, followed by cleavage and release., Results: In this study, we used MpA (MKQLEDKIEELLSKAAMKQLEDKIEELLSK) as a second class of self-assembling peptide in the cSAT scheme. This scheme utilizes low salt concentration to keep the fusion protein soluble, while eliminating insoluble cellular matters by centrifugation. Salt then triggers MpA-mediated self-aggregation of the fusion, removing soluble background host cell proteins. Finally, intein-mediated cleavage releases the target protein into solution. As a proof-of-concept, we successfully purified four proteins and peptides (human growth hormone, 22.1 kDa; LCB3, 7.7 kDa; SpyCatcherΔN-ELP-SpyCatcherΔN, 26.2 kDa; and xylanase, 45.3 kDa) with yields ranging from 12 to 87 mg/L. This was comparable to the classical His-tag method both in yield and purity (72-97%), but without the His-tag. By using a further two-step column purification process that included ion-exchange chromatography and size-exclusion chromatography, the purity was increased to over 99%., Conclusion: Our results demonstrate that a salt-inducible self-assembling peptide can serve as a controllable aggregating tag, which might be advantageous in applications where soluble expression of the target protein is preferred. This work also demonstrates the potential and advantages of utilizing salt inducible self-assembling peptides for protein separation., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
45. A Convenient Self-Removing Affinity Tag Method for the Simple Purification of Tagless Recombinant Proteins.
- Author
-
Prabhala SV, Mayone SA, Moody NM, Kanu CB, and Wood DW
- Subjects
- Humans, Recombinant Proteins genetics, Recombinant Proteins chemistry, Protein Splicing, Inteins, Nostoc genetics, Nostoc chemistry
- Abstract
In this work, we describe a novel self-cleaving affinity tag technology based on a highly modified split-intein cleaving element. In this system, which has recently been commercialized by Protein Capture Science, LLC under the name iCapTag
TM , the N-terminal segment of an engineered split intein is covalently immobilized onto a capture resin, while the smaller C-terminal intein segment is fused to the N-terminus of the desired target protein. The tagged target can then be expressed in an appropriate expression system, without concern for premature intein cleaving. During the purification, strong binding between the intein segments effectively captures the tagged target onto the capture resin while simultaneously generating a cleaving-competent intein complex. After unwanted impurities are washed from the resin, cleavage of the target protein is initiated by a shift of the buffer pH from 8.5 to 6.2. As a result, the highly purified tagless target protein is released from the column in the elution step. Alternately, the resin beads can be added directly to cell culture broth or lysate, allowing capture, purification and cleavage of the tagless target protein using a column-free format. These methods result in highly pure tagless target protein in a single step, and can thereby accelerate characterization and functional studies. In this work we demonstrate the single step purification of streptokinase, a fibrinolytic agent, and an engineered recombinant human hemoglobin 1.1 (rHb1.1). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression of high-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Basic Protocol 2: Purification of high-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform Alternate Protocol 1: Expression of low-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Alternate Protocol 2: Purification of low-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)- Published
- 2023
- Full Text
- View/download PDF
46. Switchable inteins for conditional protein splicing.
- Author
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Di Ventura, Barbara and Mootz, Henning D.
- Subjects
- *
INTEINS , *CHEMICAL biology , *OPTOGENETICS , *PROTEINS , *SMALL molecules - Abstract
Synthetic biologists aim at engineering controllable biological parts such as DNA, RNA and proteins in order to steer biological activities using external inputs. Proteins can be controlled in several ways, for instance by regulating the expression of their encoding genes with small molecules or light. However, post-translationally modifying pre-existing proteins to regulate their function or localization leads to faster responses. Conditional splicing of internal protein domains, termed inteins, is an attractive methodology for this purpose. Here we discuss methods to control intein activity with a focus on those compatible with applications in living cells. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
47. Production of antimicrobial peptide arasin-like Sp in Escherichia coli via an ELP-intein self-cleavage system
- Author
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Xiu, Li, Yu, Jiang, and Ying, Lin
- Subjects
Recombinant Fusion Proteins ,Escherichia coli ,Animals ,Bioengineering ,General Medicine ,Applied Microbiology and Biotechnology ,Antimicrobial Peptides ,Antimicrobial Cationic Peptides ,Bacillus subtilis ,Inteins ,Biotechnology - Abstract
Antibiotic resistance is a major public health threat to both humans and animals. There is an urgent need for antimicrobial agents with novel modes of action. Antimicrobial peptides (AMPs) with broad-spectrum antimicrobial activity become the ideal alternative to traditional antibiotics. Here, the ELP-intein self-cleavage system was used to produce antimicrobial peptide arasin-likeSp in Escherichia coli. The tagged target protein (ELP-intein-arasin-likeSp) was mainly expressed in soluble, separated from cell lysates by the inverse transition cycling (ITC), and the arasin-likeSp was further purified by the self-cleavage of intein and the second round of ITC. The final yield of arasin-likeSp was about 3.56 mg/L. Purified arasin-likeSp exhibited significant antibacterial activities against the Gram-positive Bacillus subtilis and Gram-negative Vibrio harveyi bacteria. FE-SEM and PI staining analysis revealed that the arasin-likeSp treatment altered the morphology and membrane permeability of Bacillus subtilis and Vibrio harveyi. Collectively, these data suggest that arasin-likeSp is a candidate AMP for effective inhibition of Vibrio harveyi, a significant bacterial pathogen infecting marine fish and invertebrates. The ELP-intein self-cleavage system described here is a low-cost, simple and potential method for producing antimicrobial peptides, which lays foundations for the large-scale production of antimicrobial peptides in the future.
- Published
- 2022
48. Long-range regulation of p53 DNA binding by its intrinsically disordered N-terminal transactivation domain.
- Author
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Krois, Alexander S., Dyson, H. Jane, and Wright, Peter E.
- Subjects
- *
DNA-binding proteins , *TETRAMERS (Oligomers) , *INTEINS , *TRANSCRIPTION factors , *DNA synthesis - Abstract
Atomic resolution characterization of the full-length p53 tetramer has been hampered by its size and the presence of extensive intrinsically disordered regions at both the N and C termini. As a consequence, the structural characteristics and dynamics of the disordered regions are poorly understood within the context of the intact p53 tetramer. Here we apply trans-intein splicing to generate segmentally 15N-labeled full-length p53 constructs in which only the resonances of the N-terminal transactivation domain (NTAD) are visible in NMR spectra, allowing us to observe this region of p53 with unprecedented detail within the tetramer. The N-terminal region is dynamically disordered in the full-length p53 tetramer, fluctuating between states in which it is free and fully exposed to solvent and states in which it makes transient contacts with the DNA-binding domain (DBD). Chemical-shift changes and paramagnetic spin-labeling experiments reveal that the amphipathic AD1 and AD2 motifs of the NTAD interact with the DNA-binding surface of the DBD through primarily electrostatic interactions. Importantly, this interaction inhibits binding of nonspecific DNA to the DBD while having no effect on binding to a specific p53 recognition element. We conclude that the NTAD: DBD interaction functions to enhance selectivity toward target genes by inhibiting binding to nonspecific sites in genomic DNA. This work provides some of the highest-resolution data on the disordered N terminus of the nearly 180-kDa full-length p53 tetramer and demonstrates a regulatory mechanism by which the N terminus of p53 transiently interacts with the DBD to enhance target site discrimination. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
49. Intermolecular disulfide bonds between unpaired cysteines retard the C-terminal trans-cleavage of Npu DnaE.
- Author
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Xu, Yanran, Zhang, Lei, Ma, Buyong, Hu, Lifu, Lu, Huili, Dou, Tonglu, Chen, Junsheng, and Zhu, Jianwei
- Subjects
- *
DISULFIDES , *CYSTEINE , *INTEINS , *C-terminal binding proteins , *GENETIC mutation , *CONFORMATIONAL analysis - Abstract
Npu DnaE is a naturally occurred split intein possessing robust trans-splicing activity and could be engineered to perform rapid C-terminal cleavage module by a single mutation D118G. Unfortunately, however, for this modified selfcleaving module, reducing agents were needed to trigger the rapid cleavage, which prevents the utilization in purification of disulfide bonds containing recombinant proteins. In this study, we demonstrated that the unpaired cysteine residues in Npu DnaE tend to form disulfide bonds, and contributed to the reduction of the cleavage under non-reducing conditions. This redox trap can be disrupted by site-directed mutation of these unpaired cysteines. The results further indicated that the position 28 and 59 may play certain roles in the correct folding of the active conformation. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
50. Conditional Alternative Protein Splicing Promoted by Inteins from Haloquadratum walsbyi
- Author
-
Vaishnavi R. Yalala, Abigeal K. Lynch, and Kenneth V. Mills
- Subjects
Halobacteriaceae ,Minichromosome Maintenance Proteins ,Archaeal Proteins ,Exteins ,Escherichia coli ,Protein Splicing ,Protein Precursors ,Peptides ,Biochemistry ,Article ,Inteins - Abstract
Protein splicing is a post-translational process by which an intervening protein, or intein, catalyzes its own excision from flanking polypeptides, or exteins, coupled to extein ligation. Four inteins interrupt the MCM helicase of the halophile Haloquadratum walsbyi, two of which are mini-inteins that lack a homing endonuclease. Both inteins can be over-expressed in E. coli and purified as unspliced precursors; splicing can be induced in vitro on incubation with salt. However, one intein can splice at 0.5 M NaCl in vitro, whereas the other splices efficiently only above 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.
- Published
- 2022
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