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A journey down to hell: new thermostable protein-tags for biotechnology at high temperatures

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  • 12th International Congress on Extremophiles
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Abstract

The specific labelling of proteins in recent years has made use of self-labelling proteins, such as the SNAP-tag® and the Halotag®. These enzymes, by their nature or suitably engineered, have the ability to specifically react with their respective substrates, but covalently retaining a part of them in the catalytic site upon reaction. This led to the synthesis of substrates conjugated with, e.g., fluorophores (proposing them as alternatives to fluorescent proteins), but also with others chemical groups, for numerous biotechnological applications. Recently, a mutant of the OGT from Saccharolobus solfataricus (H5) very stable to high temperatures and in the presence of physical and chemical denaturing agents has been proposed as a thermostable SNAP-tag® for in vivo and in vitro harsh reaction conditions. Here, we show two new thermostable OGTs from Thermotoga neapolitana and Pyrococcus furiosus, which, respectively, display a higher catalytic activity and thermostability respect to H5, proposing them as alternatives for in vivo studies in these extreme model organisms.

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References

  • Aliye N, Fabbretti A, Lupidi G, Tsekoa T, Spurio R (2015) Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics. Appl Microbiol Biotechnol 99:1205–1216

    CAS  PubMed  Google Scholar 

  • Aoki K, Natsume A (2019) Overview of DNA methylation in adult diffuse gliomas. Brain Tumor Pathol 36:84–91

    PubMed  Google Scholar 

  • Ashby MC, Ibaraki K, Henley JM (2004) It’s green outside: tracking cell surface proteins with pH-sensitive GFP. Trends Neurosci 27:257–261

    CAS  PubMed  Google Scholar 

  • Belkin S, Wirsen CO, Jannasch HW (1986) A new sulfur-reducing, extremely thermophilic eubacterium from a submarine thermal vent. App Environ Microbiol 51:1180–1185

    CAS  Google Scholar 

  • Campbell TN, Choy FYM (2000) The effect of pH on green fluorescent protein: a brief review. Mol Biol Today 2:1–4

    Google Scholar 

  • Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 5148:802–805

    Google Scholar 

  • Conners SB, Mongodin EF, Johnson MR, MonteroCI Nelson KE, Kelly RM (2006) Microbial biochemistry, physiology, and biotechnology of hyperthermophilic Thermotoga species. FEMS Microb Rev 30:872–905

    CAS  Google Scholar 

  • Coulter R, Blandino M, Tomlinson JM, Pauly GT, Krajewska M, Moschel RC, Peterson LA, Pegg AE, Spratt TE (2007) Differences in the rate of repair of O 6-alkylguanines in different sequence contexts by O 6-alkylguanine-DNA alkyltransferase. Chem Res Toxicol 20:1966–1971

    CAS  PubMed  Google Scholar 

  • d’Ippolito G, Dipasquale L, Vella FM, Romano I, Gambacorta A, Cutignano A, Fontana A (2010) Hydrogen metabolism in the extreme thermophile Thermotoga neapolitana. Int J Hydrog En 35:2290–2295

    Google Scholar 

  • d’Ippolito G, Dipasquale L, Fontana A (2014) Recycling of carbon dioxide and acetate as lactic acid by the hydrogen-producing bacterium Thermotoga neapolitana. Chemsuschem 7:2678–2683

    PubMed  Google Scholar 

  • Daniels DS, Mol CD, Arvai AS, Kanugula S, Pegg AE, Tainer JA (2000) Active and alkylated human AGT structures: a novel zinc site, inhibitor and extrahelical base binding. EMBO J 19:1719–1730

    CAS  PubMed  PubMed Central  Google Scholar 

  • Daniels DS, Woo TT, Luu KX, Noll DM, Clarke ND, Pegg AE, Tainer JA (2004) DNA binding and nucleotide flipping by the human DNA repair protein AGT. Nat Struct Mol Biol 11:714–720

    CAS  PubMed  Google Scholar 

  • Del Prete S, Merlo R, Valenti A, Mattossovich R, Rossi M, Carginale M, Supuran CT, Perugino G, Capasso C (2019) Thermostability enhancement of the α-carbonic anhydrase from Sulfurihydrogenibium yellowstonense by using the anchoring-and-selflabelling-protein-tag system (ASLtag). J Enz Inhib Med Chem 34:946–954

    Google Scholar 

  • Dipasquale L, d’Ippolito G, Fontana A (2014) Capnophilic lactic fermentation and hydrogen synthesis by Thermotoga neapolitana: an unexpected deviation from the dark fermentation model. Int J Hydrog En 39:4857–4862

    CAS  Google Scholar 

  • Donaldson T, Iozzino L, Deacon LJ, Billones H, Ausili A, D’Auria S, Dattelbaum JD (2017) Engineering a switch-based biosensor for arginine using a Thermotoga maritima periplasmic binding protein. An Biochem 525:60–66

    CAS  Google Scholar 

  • Fang Q, Kanugula S, Pegg AE (2005) Function of domains of human O 6-alkyl-guanine-DNA alkyltransferase. Biochemistry 44:15396–15405

    CAS  PubMed  Google Scholar 

  • Fiala G, Stetter KO (1986) Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100 °C. Arch Microb 145:56–61

    CAS  Google Scholar 

  • Fink M, Trunk S, Hall M, Schwab H, Steiner K (2016) Engineering of TM1459 from Thermotoga maritima for increased oxidative alkene cleavage activity. Front Microb. https://doi.org/10.3389/fmicb.2016.01511

    Article  Google Scholar 

  • Gautier A, Juillerat A, Heinis C, Corrêa IR Jr, Kindermann M, Beaufils F, Johnsson K (2008) An engineered protein-tag for multiprotein labeling in living cells. Chem Biol 15:128–136

    CAS  PubMed  Google Scholar 

  • Gronemeyer T, Chidley C, Juillerat A, Heinis C, Johnsson K (2006) Directed evolution of O 6-alkylguanine-DNA alkyltransferase for applications in protein labeling. Prot Eng Des Sel 19:309–316

    CAS  Google Scholar 

  • Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP (2009) RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139:945–956

    CAS  PubMed  PubMed Central  Google Scholar 

  • Han D, Xu Z (2017) Development of a pyrE-based selective system for Thermotoga sp. strain RQ7. Extremophiles 21:297–306

    CAS  PubMed  Google Scholar 

  • Hashimoto H, Inouel T, Nishioka M, Fujiwara S, Takagi M, Imanaka T, Kai Y (1999) Hyperthermostable protein structure maintained by intra and inter-helix ion-pairs in archaeal 06-methylguanine-DNA methyltransferase. J Mol Biol 292:707–716

    CAS  PubMed  Google Scholar 

  • Hinner MJ, Johnsson K (2010) How to obtain labeled proteins and what to do with them. Curr Opin Biotechnol 21:766–776

    CAS  PubMed  Google Scholar 

  • Ishiguro K, Shyam K, Penketh PG, Sartorelli AC (2008) Development of an O 6-alkyl-guanine-DNA alkyltransferase assay based on covalent transfer of the benzyl moiety from [benzene-3H]O 6-benzylguanine to the protein. Anal Biochem 383:44–51

    CAS  PubMed  PubMed Central  Google Scholar 

  • Jannasch HW, Huber R, Belkin S, Stetter KO (1988) Thermotoga neapolitana sp. nov. of the extremely thermophilic, eubacterial genus Thermotoga. Arch Microbiol 150:103–104

    Google Scholar 

  • Juillerat A, Gronemeyer T, Keppler A, Gendreizig S, Pick H, Vogel H, Johnsson K (2003) Directed evolution of O 6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo. Chem Biol 10(4):313–317

    CAS  PubMed  Google Scholar 

  • Kengen SWM (2017) Pyrococcus furiosus, 30 years on. Microb Biotechnol 10:1441–1444

    PubMed  PubMed Central  Google Scholar 

  • Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel Johnsson K (2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21:86–89

    CAS  PubMed  Google Scholar 

  • Keppler A, Pick H, Arrivoli C, Vogel H, Johnsson K (2004) Labeling of fusion proteins with synthetic fluorophores in live cells. Proc Natl Acad Sci U S A 10:9955–9959

    Google Scholar 

  • Leclere MM, Nishioka M, Yuasa T, Fujiwara S, Takagi M, Imanaka T (1998) The O 6-methylguanine-DNA methyltransferase from the hyperthermophilic archaeon Pyrococcus sp. KOD1: a thermostable repair enzyme. Mol Gen Genet 258:69–77

    CAS  PubMed  Google Scholar 

  • Lo-Gullo G, Mattossovich R, Perugino G, La Teana A, Londei P, Benelli D (2019) Optimization of an in vitro transcription/translation system based on Sulfolobus solfataricus cell lysate. Archaea 1:1–10

    Google Scholar 

  • Lundberg KS, Shoemaker DD, Adams MWW, Short JM, Sorge JA, Mathur EJ (1991) High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108:1–6

    CAS  PubMed  Google Scholar 

  • Luu KX, Kanugula S, Pegg AE, Pauly GT, Moschel RC (2002) Repair of oligodeoxyribonucleotides by O 6-alkylguanine-DNA alkyltransferase. Biochemistry 41:8689–8697

    CAS  PubMed  Google Scholar 

  • Merlo R, Del Prete S, Valenti A, Mattossovich R, Carginale V, Supuran CT, Capasso C, Perugino G (2019) An AGT-based protein-tag system for the labelling and surface immobilization of enzymes on E. coli outer membrane. J Enz Inhib Med Chem 34:490–499

    CAS  Google Scholar 

  • Miggiano R, Casazza V, Garavaglia S, Ciaramella M, Perugino G, Rizzi M, Rossi F (2013) Biochemical and structural studies of the Mycobacterium tuberculosis O 6-methylguanine methyltransferase and mutated variants. J Bacteriol 195:2728–2736

    CAS  PubMed  PubMed Central  Google Scholar 

  • Miggiano R, Valenti A, Rossi F, Rizzi M, Perugino G, Ciaramella M (2017) Every OGT is illuminated … by fluorescent and synchrotron lights. Int J Mol Sci 18:2613–2631

    PubMed Central  Google Scholar 

  • Mishina Y, Duguid EM, He C (2006) Direct reversal of DNA alkylation damage. Chem Rev 106:215–232

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mollwitz B, Brunk E, Schmitt S, Pojer F, Bannwarth M, Schiltz M, Rothlisberger U, Johnsson K (2012) Directed evolution of the suicide protein O 6-alkylguanine-DNA alkyltransferase for Increased reactivity results in an alkylated protein with exceptional stability. Biochemistry 51:986–994

    CAS  PubMed  Google Scholar 

  • Morrone C, Miggiano R, Serpe M, Massarotti A, Valenti A, del Monaco G, Rossi M, Rossi F, Rizzi M, Perugino G, Ciaramella M (2017) Interdomain interactions rearrangements control the reaction steps of a thermostable DNA alkyltransferase. Biochem Biophys Acta 1861:86–96

    CAS  Google Scholar 

  • Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2:2212–2221

    CAS  PubMed  Google Scholar 

  • Nishikori S, Shiraki K, Fujiwara S, Imanaka T, Takagi M (2005) Unfolding mechanism of a hyperthermophilic protein O 6-methylguanine-DNA methyltransferase. Biophys Chem 116:97–104

    CAS  PubMed  Google Scholar 

  • Pegg AE (2011) Multifaceted roles of alkyltransferase and related proteins in DNA repair, DNA damage, resistance to chemotherapy, and research tools. Chem Res Toxicol 24:618–639

    CAS  PubMed  PubMed Central  Google Scholar 

  • Pegg AE, Goodtzova K, Loktionova NA, Kanugula S, Pauly GT, Moschel RC (2001) Inactivation of human O 6-alkylguanine-DNA alkyltransferase by modified oligodeoxyribonucleotides containing O 6-benzylguanine. J Pharmacol Exp Ther 296:958–965

    CAS  PubMed  Google Scholar 

  • Perugino G, Vettone V, Illiano G, Valenti A, Ferrara MC, Rossi M, Ciaramella M (2012) Activity and regulation of archaeal DNA alkyltransferase: conserved protein involved in repair of DNA alkylation damage. J Biol Chem 287:4222–4231

    CAS  PubMed  Google Scholar 

  • Perugino G, Miggiano R, Serpe M, Vettone A, Valenti A, Lahiri S, Rossi F, Rossi M, Rizzi M, Ciaramella M (2015) Structure-function relationships governing activity and stability of a DNA alkylation damage repair thermostable protein. Nucleic Acids Res 43:8801–8816

    CAS  PubMed  PubMed Central  Google Scholar 

  • Pradhan N, Dipasquale L, d’Ippolito G, Panico A, Lens PNL, Esposito G, Fontana A (2015) Hydrogen production by the thermophilic bacterium Thermotoga neapolitana. Int J Mol Sci 16:12578–12600

    CAS  PubMed  PubMed Central  Google Scholar 

  • Pradhan N, Dipasquale L, d’Ippolito G, Fontana A, Panico A, Lens PNL, Pirozzi F, Esposito G (2016) Kinetic modeling of fermentative hydrogen production by Thermotoga neapolitana. Int J Hydrog Energy 41:4931–4940

    CAS  Google Scholar 

  • Pradhan N, Dipasquale L, d’Ippolito G, Panico A, Lens PNL, Esposito G, Fontana A (4359T) Hydrogen and lactic acid synthesis by the wild-type and a laboratory strain of the hyperthermophilic bacterium Thermotoga neapolitana DSMZ 4359T under capnophilic lactic fermentation conditions. Int J Hydrog Energy 42:16023–16030

    CAS  Google Scholar 

  • Samuelson P, Gunneriusson E, Nygren PA, Ståhl S (2002) Display of proteins on bacteria. J Biotechnol 96:129–154

    CAS  PubMed  Google Scholar 

  • Serpe M, Forenza C, Adamo A, Russo N, Perugino G, Ciaramella M, Valenti A (2019) The DNA Alkylguanine DNA Alkyltransferase-2 (AGT-2) of Caenorhabditis Elegans is involved in meiosis and early development under physiological conditions. Sci Rep 9:6889

    PubMed  PubMed Central  Google Scholar 

  • Skorvaga M, Raven NDH, Margison GP (1998) Thermostable archaeal O 6-alkylguanine-DNA alkyltransferases. Proc Natl Acad Sci USA 95:6711–6715

    CAS  PubMed  Google Scholar 

  • Sun G, Zhao L, Zhong R, Peng Y (2018) The specific role of O 6-methylguanine-DNA methyltransferase inhibitors in cancer chemotherapy. Future Med Chem 10:1971–2199

    CAS  PubMed  Google Scholar 

  • Terns RM, Terns MP (2013) The RNA- and DNA-targeting CRISP-Cas immune systems of Pyrococcus furiosus. Biochem Soc Trans 41:1416–1421

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544

    CAS  PubMed  Google Scholar 

  • Tubbs JL, Pegg AE, Tainer JA (2007) DNA binding, nucleotide flipping, and the helix-turn-helix motif in base repair by O 6-alkylguanine-DNA alkyltransferase and its implications for cancer chemotherapy. DNA Repair 6:1100–1115

    CAS  PubMed  PubMed Central  Google Scholar 

  • Vettone A, Serpe M, Hidalgo A, Berenguer J, del Monaco G, Valenti A, Rossi M, Ciaramella M, Perugino G (2016) A novel thermostable protein-tag: optimization of the Sulfolobus solfataricus DNA-alkyl-transferase by protein engineering. Extremophiles 20:1–13

    CAS  PubMed  Google Scholar 

  • Visone V, Han W, Perugino G, del Monaco G, She Q, Rossi M, Valenti A, Ciaramella M (2017) In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag. PLoS ONE 12:e0185791

    PubMed  PubMed Central  Google Scholar 

  • Yang CG, Garcia K, He C (2009) Damage detection and base flipping in direct DNA alkylation repair. ChemBioChem 10:417–423

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang J, Shi H, Xu L, Zhu X, Li X (2015) Site-directed mutagenesis of a hyperthermophilic endoglucanase Cel12B from Thermotoga maritima based on rational design. PLoS ONE. https://doi.org/10.1371/journal.pone.0133824

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by Fondazione CARIPLO (Ricerca biomedical condotta da giovani ricercatori, project 2016-0604).

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  1. Rosanna Mattossovich and Rosa Merlo equally contributed to the present work.

Authors and Affiliations

  1. Institute of Biosciences and BioResources, National Council of Research of Italy, Via P. Castellino 111, 80131, Naples, Italy

    Rosanna Mattossovich, Rosa Merlo, Anna Valenti & Giuseppe Perugino

  2. Institute of Biomolecular Chemistry, National Council of Research of Italy, Via Campi Flegrei, 34, 80078, Pozzuoli, NA, Italy

    Angelo Fontana & Giuliana d’Ippolito

  3. Departments of Biochemistry and Molecular Biology, Genetics, and Microbiology, University of Georgia, Athens, GA, USA

    Michael P. Terns & Elizabeth A. Watts

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  1. Rosanna Mattossovich
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Correspondence to Giuseppe Perugino.

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Communicated by M. Moracci.

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This manuscript is part of a special issue of Extremophiles journal for the 12th International Congress of Extremophiles (Extremophiles 2018) that was held on 16–20 September 2018 in Ischia, Naples, Italy.

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Mattossovich, R., Merlo, R., Fontana, A. et al. A journey down to hell: new thermostable protein-tags for biotechnology at high temperatures. Extremophiles 24, 81–91 (2020). https://doi.org/10.1007/s00792-019-01134-3

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