Bec G, Ennifar E
switchSENSE Technology for Analysis of DNA Polymerase Kinetics Book Chapter
In: Rederstorff, M (Ed.): vol. 2247, pp. 145-153, Springer Protocols, Humana Press, New York, NY, Small Non-Coding RNAs, 2021.
Abstract | BibTeX | Tags: Biosensor, DNA polymerase, ENNIFAR, HIV reverse transcriptase, Kinetics, switchSENSE technology, Unité ARN
@inbook{Bec2021,
title = {switchSENSE Technology for Analysis of DNA Polymerase Kinetics },
author = {G Bec and E Ennifar
},
editor = {M Rederstorff},
year = {2021},
date = {2021-01-01},
journal = {Methods in Molecular Biology },
volume = {2247},
pages = {145-153},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
edition = {Small Non-Coding RNAs},
series = {Methods in Molecular Biology},
abstract = {The switchSENSE technology is a recent approach based on surface sensor chips for the analysis of interactions of macromolecules. The technology relies on electro-switchable DNA nanolevers tethered at one end on a gold surface via a sulfur linker and labeled with a Cy3 dye on the other end. The switchSENSE approach is effective in the determination of a large panel of biophysical parameters such as binding kinetics, dissociation constant, hydrodynamic radius, or melting temperature. In addition, it can also give access to some enzymatic data such as nuclease or polymerase activity. Here we describe a DNA polymerase assay that allows retrieving, in a single experimental set, association and dissociation rates, as well as the catalytic rate of the enzyme. },
keywords = {Biosensor, DNA polymerase, ENNIFAR, HIV reverse transcriptase, Kinetics, switchSENSE technology, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Schenckbecher E, Bec G, Sakamoto T, Meyer B, Ennifar E
Biophysical Studies of the Binding of Viral RNA with the 80S Ribosome Using switchSENSE Journal Article
In: Methods Mol Biol, vol. 2263, pp. 341-350, 2021, ISBN: 33877606, (1940-6029 (Electronic) 1064-3745 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Biophysics, ENNIFAR, Kinetics, Ribosome, RNA, switchSENSE, Unité ARN
@article{,
title = {Biophysical Studies of the Binding of Viral RNA with the 80S Ribosome Using switchSENSE},
author = {E Schenckbecher and G Bec and T Sakamoto and B Meyer and E Ennifar},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33877606},
doi = {10.1007/978-1-0716-1197-5_15},
isbn = {33877606},
year = {2021},
date = {2021-01-01},
journal = {Methods Mol Biol},
volume = {2263},
pages = {341-350},
abstract = {Translation initiation, in both eukaryotes and bacteria, requires essential elements such as mRNA, ribosome, initiator tRNA, and a set of initiation factors. For each domain of life, canonical mechanisms and signals are observed to initiate protein synthesis. However, other initiation mechanism can be used, especially in viral mRNAs. Some viruses hijack cellular machinery to translate some of their mRNAs through a noncanonical initiation pathway using internal ribosome entry site (IRES), a highly structured RNAs which can directly recruit the ribosome with a restricted set of initiation factors, and in some cases even without cap and initiator tRNA. In this chapter, we describe the use of biosensors relying on electro-switchable nanolevers using the switchSENSE((R)) technology, to investigate kinetics of the intergenic (IGR) IRES of the cricket paralysis virus (CrPV) binding to 80S yeast ribosome. This study provides a proof of concept for the application of this method on large complexes.},
note = {1940-6029 (Electronic)
1064-3745 (Linking)
Journal Article},
keywords = {Biophysics, ENNIFAR, Kinetics, Ribosome, RNA, switchSENSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Benincasa Monica, Pacor Sabrina, Wu Wei, Prato Maurizio, Bianco Alberto, Gennaro Renato
Antifungal activity of amphotericin B conjugated to carbon nanotubes Journal Article
In: ACS nano, vol. 5, no. 1, pp. 199–208, 2011, ISSN: 1936-086X.
Abstract | Links | BibTeX | Tags: Amphotericin B, Antifungal Agents, Candida, carbon, Cell Membrane, Deoxycholic Acid, Drug Design, Drug Resistance, Fungal, Humans, I2CT, Jurkat Cells, Kinetics, Membrane Potentials, Nanotubes, Team-Bianco
@article{benincasa_antifungal_2011,
title = {Antifungal activity of amphotericin B conjugated to carbon nanotubes},
author = {Monica Benincasa and Sabrina Pacor and Wei Wu and Maurizio Prato and Alberto Bianco and Renato Gennaro},
doi = {10.1021/nn1023522},
issn = {1936-086X},
year = {2011},
date = {2011-01-01},
journal = {ACS nano},
volume = {5},
number = {1},
pages = {199--208},
abstract = {Amphotericin B (AMB) has long been considered the most effective drug in the treatment of serious invasive fungal infections. There are, however, major limitations to its use, due to several adverse effects, including acute infusional reactions and, most relevant, a dose-dependent nephrotoxicity. At least some of these effects are attributed to the aggregation of AMB as a result of its poor water solubility. To overcome this problem, reformulated versions of the drug have been developed, including a micellar dispersion of AMB with sodium deoxycholate (AMBD), its encapsulation into liposomes, or its incorporation into lipidic complexes. The development of nanobiotechnologies provides novel potential drug delivery systems that make use of nanomaterials such as functionalized carbon nanotubes (f-CNTs), which are emerging as an innovative and efficient tool for the transport and cellular translocation of therapeutic molecules. In this study, we prepared two conjugates between f-CNTs and AMB. The antifungal activity of these conjugates was tested against a collection of reference and clinical fungal strains, in comparison to that of AMB alone or AMBD. Measured minimum inhibition concentration (MIC) values for f-CNT-AMB conjugates were either comparable to or better than those displayed by AMB and AMBD. Furthermore, AMBD-resistant Candida strains were found to be susceptible to f-CNT-AMB 1. Additional studies, aimed at understanding the mechanism of action of the conjugates, suggest a nonlytic mechanism, since the compounds show a major permeabilizing effect on the tested fungal strains only after extended incubation. Interestingly, the f-CNT-AMB 1 does not show any significant toxic effect on Jurkat cells at antifungal concentrations.},
keywords = {Amphotericin B, Antifungal Agents, Candida, carbon, Cell Membrane, Deoxycholic Acid, Drug Design, Drug Resistance, Fungal, Humans, I2CT, Jurkat Cells, Kinetics, Membrane Potentials, Nanotubes, Team-Bianco},
pubstate = {published},
tppubtype = {article}
}
Whisstock James C, Silverman Gary A, Bird Phillip I, Bottomley Stephen P, Kaiserman Dion, Luke Cliff J, Pak Stephen C, Reichhart Jean-Marc, Huntington James A
Serpins flex their muscle: II. Structural insights into target peptidase recognition, polymerization, and transport functions Journal Article
In: J. Biol. Chem., vol. 285, no. 32, pp. 24307–24312, 2010, ISSN: 1083-351X.
Abstract | Links | BibTeX | Tags: Animals, Biological, Biological Transport, Biophysics, Catalytic Domain, Hormones, Humans, Kinetics, M3i, Models, Peptide Hydrolases, Protein Binding, Protein Conformation, Protein Structure, reichhart, Serpins, Substrate Specificity, Tertiary, Thrombin
@article{whisstock_serpins_2010,
title = {Serpins flex their muscle: II. Structural insights into target peptidase recognition, polymerization, and transport functions},
author = {James C Whisstock and Gary A Silverman and Phillip I Bird and Stephen P Bottomley and Dion Kaiserman and Cliff J Luke and Stephen C Pak and Jean-Marc Reichhart and James A Huntington},
doi = {10.1074/jbc.R110.141408},
issn = {1083-351X},
year = {2010},
date = {2010-08-01},
journal = {J. Biol. Chem.},
volume = {285},
number = {32},
pages = {24307--24312},
abstract = {Inhibitory serpins are metastable proteins that undergo a substantial conformational rearrangement to covalently trap target peptidases. The serpin reactive center loop contributes a majority of the interactions that serpins make during the initial binding to target peptidases. However, structural studies on serpin-peptidase complexes reveal a broader set of contacts on the scaffold of inhibitory serpins that have substantial influence on guiding peptidase recognition. Structural and biophysical studies also reveal how aberrant serpin folding can lead to the formation of domain-swapped serpin multimers rather than the monomeric metastable state. Serpin domain swapping may therefore underlie the polymerization events characteristic of the serpinopathies. Finally, recent structural studies reveal how the serpin fold has been adapted for non-inhibitory functions such as hormone binding.},
keywords = {Animals, Biological, Biological Transport, Biophysics, Catalytic Domain, Hormones, Humans, Kinetics, M3i, Models, Peptide Hydrolases, Protein Binding, Protein Conformation, Protein Structure, reichhart, Serpins, Substrate Specificity, Tertiary, Thrombin},
pubstate = {published},
tppubtype = {article}
}
Burnouf D. Y., Wagner J. E.
Kinetics of deoxy-CTP incorporation opposite a dG-C8-N-2-aminofluorene adduct by a high-fidelity DNA polymerase Journal Article
In: J Mol Biol, vol. 386, no. 4, pp. 951-61, 2009, (1089-8638 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | BibTeX | Tags: Adducts, Bacillus, Catalytic, Cytidine, Deoxyguanosine/*metabolism, DNA, DNA-Directed, Domain, DUMAS, Elements, Fluorenes/*metabolism, Guanine, Kinetics, Oligonucleotides/metabolism, Phosphorothioate, Polymerase/*metabolism, Specificity, stearothermophilus/enzymology, Substrate, Titrimetry, Triphosphate/*metabolism
@article{,
title = {Kinetics of deoxy-CTP incorporation opposite a dG-C8-N-2-aminofluorene adduct by a high-fidelity DNA polymerase},
author = { D. Y. Burnouf and J. E. Wagner},
year = {2009},
date = {2009-01-01},
journal = {J Mol Biol},
volume = {386},
number = {4},
pages = {951-61},
abstract = {The model carcinogen N-2-acetylaminofluorene covalently binds to the C8 position of guanine to form two adducts, the N-(2'-deoxyguanosine-8-yl)-aminofluorene (G-AF) and the N-2-(2'-deoxyguanosine-8-yl)-acetylaminofluorene (G-AAF). Although they are chemically closely related, their biological effects are strongly different and they are processed by different damage tolerance pathways. G-AF is bypassed by replicative and high-fidelity polymerases, while specialized polymerases ensure synthesis past of G-AAF. We used the DNA polymerase I fragment of a Bacillus stearothermophilus strain as a model for a high-fidelity polymerase to study the kinetics of incorporation of deoxy-CTP (dCTP) opposite a single G-AF. Pre-steady-state kinetic experiments revealed a drastic reduction in dCTP incorporation performed by the G-AF-modified ternary complex. Two populations of these ternary complexes were identified: (i) a minor productive fraction (20%) that readily incorporates dCTP opposite the G-AF adduct with a rate similar to that measured for the adduct-free ternary complexes and (ii) a major fraction of unproductive complexes (80%) that slowly evolve into productive ones. In the light of structural data, we suggest that this slow rate reflects the translocation of the modified base within the active site, from the pre-insertion site into the insertion site. By making this translocation rate limiting, the G-AF lesion reveals a novel kinetic step occurring after dNTP binding and before chemistry.},
note = {1089-8638 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Adducts, Bacillus, Catalytic, Cytidine, Deoxyguanosine/*metabolism, DNA, DNA-Directed, Domain, DUMAS, Elements, Fluorenes/*metabolism, Guanine, Kinetics, Oligonucleotides/metabolism, Phosphorothioate, Polymerase/*metabolism, Specificity, stearothermophilus/enzymology, Substrate, Titrimetry, Triphosphate/*metabolism},
pubstate = {published},
tppubtype = {article}
}
Wilkins Sarah E, Hyvärinen Jaana, Chicher Johana, Gorman Jeffrey J, Peet Daniel J, Bilton Rebecca L, Koivunen Peppi
Differences in hydroxylation and binding of Notch and HIF-1alpha demonstrate substrate selectivity for factor inhibiting HIF-1 (FIH-1). Journal Article
In: The international journal of biochemistry & cell biology, vol. 41, no. 7, pp. 1563–1571, 2009, ISSN: 1878-5875 1357-2725, (Place: Netherlands).
Abstract | Links | BibTeX | Tags: alpha Subunit/*metabolism, Amino Acid Sequence, Animals, Asparagine/metabolism, Humans, Hydroxylation, Hypoxia-Inducible Factor 1, Kinetics, Mice, Mixed Function Oxygenases, Molecular Sequence Data, Notch/chemistry/*metabolism, Oxygen/metabolism, Peptides/chemistry/metabolism, PPSE, Protein Binding, Receptors, Recombinant Proteins/metabolism, Repressor Proteins/*metabolism, Substrate Specificity
@article{wilkins_differences_2009,
title = {Differences in hydroxylation and binding of Notch and HIF-1alpha demonstrate substrate selectivity for factor inhibiting HIF-1 (FIH-1).},
author = {Sarah E Wilkins and Jaana Hyvärinen and Johana Chicher and Jeffrey J Gorman and Daniel J Peet and Rebecca L Bilton and Peppi Koivunen},
doi = {10.1016/j.biocel.2009.01.005},
issn = {1878-5875 1357-2725},
year = {2009},
date = {2009-01-01},
journal = {The international journal of biochemistry & cell biology},
volume = {41},
number = {7},
pages = {1563--1571},
abstract = {FIH-1, factor inhibiting hypoxia-inducible factor-1 (HIF-1), regulates oxygen sensing by hydroxylating an asparagine within HIF-alpha. It also hydroxylates asparagines in many proteins containing ankyrin repeats, including Notch1-3, p105 and I?B?. Relative binding affinity and hydroxylation rate are crucial determinants of substrate selection and modification. We determined the contributions of substrate sequence composition and length and of oxygen concentration to the FIH-1-binding and/or hydroxylation of Notch1-4 and compared them with those for HIF-1alpha. We also demonstrated hydroxylation of two asparagines in Notch2 and 3, corresponding to Sites 1 and 2 of Notch1, by mass spectrometry for the first time. Our data demonstrate that substrate length has a much greater influence on FIH-1-dependent hydroxylation of Notch than of HIF-1alpha, predominantly through binding affinity rather than maximal reaction velocity. The K(m) value of FIH-1 for Notch1, textless 0.2 microM, is at least 250-fold lower than that of 50 microM for HIF-1alpha. Site 1 of Notch1-3 appeared the preferred site of FIH-1 hydroxylation in these substrates. Interestingly, binding of Notch4 to FIH-1 was observed with an affinity almost 10-fold lower than for Notch1-3, but no hydroxylation was detected. Importantly, we demonstrate that the K(m) of FIH-1 for oxygen at the preferred Site 1 of Notch1-3, 10-19 microM, is an order of magnitude lower than that for Site 2 or HIF-1alpha. Hence, at least during in vitro hydroxylation, Notch is likely to become efficiently hydroxylated by FIH-1 even under relatively severe hypoxic conditions, where HIF-1alpha hydroxylation would be reduced.},
note = {Place: Netherlands},
keywords = {alpha Subunit/*metabolism, Amino Acid Sequence, Animals, Asparagine/metabolism, Humans, Hydroxylation, Hypoxia-Inducible Factor 1, Kinetics, Mice, Mixed Function Oxygenases, Molecular Sequence Data, Notch/chemistry/*metabolism, Oxygen/metabolism, Peptides/chemistry/metabolism, PPSE, Protein Binding, Receptors, Recombinant Proteins/metabolism, Repressor Proteins/*metabolism, Substrate Specificity},
pubstate = {published},
tppubtype = {article}
}
Bianco Alberto, Hoebeke Johan, Godefroy Sylvie, Chaloin Olivier, Pantarotto Davide, Briand Jean-Paul, Muller Sylviane, Prato Maurizio, Partidos Charalambos D
Cationic carbon nanotubes bind to CpG oligodeoxynucleotides and enhance their immunostimulatory properties Journal Article
In: Journal of the American Chemical Society, vol. 127, no. 1, pp. 58–59, 2005, ISSN: 0002-7863.
Abstract | Links | BibTeX | Tags: Adjuvants, Animals, carbon, Cations, CpG Islands, I2CT, Immunologic, Interferon-gamma, Interleukin-6, Kinetics, Lymphocytes, Mice, Nanotubes, oligonucleotides, Surface Plasmon Resonance, Team-Bianco
@article{bianco_cationic_2005,
title = {Cationic carbon nanotubes bind to CpG oligodeoxynucleotides and enhance their immunostimulatory properties},
author = {Alberto Bianco and Johan Hoebeke and Sylvie Godefroy and Olivier Chaloin and Davide Pantarotto and Jean-Paul Briand and Sylviane Muller and Maurizio Prato and Charalambos D Partidos},
doi = {10.1021/ja044293y},
issn = {0002-7863},
year = {2005},
date = {2005-01-01},
journal = {Journal of the American Chemical Society},
volume = {127},
number = {1},
pages = {58--59},
abstract = {Functionalized cationic carbon nanotubes are able to form a stable complex with CpG ODN based on charge interaction and to increase the immunostimulatory activity of CpG motifs.},
keywords = {Adjuvants, Animals, carbon, Cations, CpG Islands, I2CT, Immunologic, Interferon-gamma, Interleukin-6, Kinetics, Lymphocytes, Mice, Nanotubes, oligonucleotides, Surface Plasmon Resonance, Team-Bianco},
pubstate = {published},
tppubtype = {article}
}
Burnouf D. Y., Olieric V., Wagner J., Fujii S., Reinbolt J., Fuchs R. P., Dumas P.
Structural and biochemical analysis of sliding clamp/ligand interactions suggest a competition between replicative and translesion DNA polymerases Journal Article
In: J Mol Biol, vol. 335, no. 5, pp. 1187-97, 2004, (0022-2836 Journal Article).
Abstract | BibTeX | Tags: *Binding, Antigen/metabolism, Bacterial/genetics, beta/*chemistry/genetics/*metabolism, Binding, Cell, coli/*enzymology, Competitive, Crystallization, DNA, DUMAS, Escherichia, Fragments/*metabolism, I/metabolism, III/metabolism, Kinetics, ligands, Models, Molecular, Nuclear, Peptide, Polymerase, Proliferating, Protein, Proteins/chemistry/metabolism, Recombinant, Replication/*genetics, Subunits
@article{,
title = {Structural and biochemical analysis of sliding clamp/ligand interactions suggest a competition between replicative and translesion DNA polymerases},
author = { D. Y. Burnouf and V. Olieric and J. Wagner and S. Fujii and J. Reinbolt and R. P. Fuchs and P. Dumas},
year = {2004},
date = {2004-01-01},
journal = {J Mol Biol},
volume = {335},
number = {5},
pages = {1187-97},
abstract = {Most DNA polymerases interact with their cognate processive replication factor through a small peptide, this interaction being absolutely required for their function in vivo. We have solved the crystal structure of a complex between the beta sliding clamp of Escherichia coli and the 16 residue C-terminal peptide of Pol IV (P16). The seven C-terminal residues bind to a pocket located at the surface of one beta monomer. This region was previously identified as the binding site of another beta clamp binding protein, the delta subunit of the gamma complex. We show that peptide P16 competitively prevents beta-clamp-mediated stimulation of both Pol IV and alpha subunit DNA polymerase activities, suggesting that the site of interaction of the alpha subunit with beta is identical with, or overlaps that of Pol IV. This common binding site for delta, Pol IV and alpha subunit is shown to be formed by residues that are highly conserved among many bacterial beta homologs, thus defining an evolutionarily conserved hydrophobic crevice for sliding clamp ligands and a new target for antibiotic drug design.},
note = {0022-2836
Journal Article},
keywords = {*Binding, Antigen/metabolism, Bacterial/genetics, beta/*chemistry/genetics/*metabolism, Binding, Cell, coli/*enzymology, Competitive, Crystallization, DNA, DUMAS, Escherichia, Fragments/*metabolism, I/metabolism, III/metabolism, Kinetics, ligands, Models, Molecular, Nuclear, Peptide, Polymerase, Proliferating, Protein, Proteins/chemistry/metabolism, Recombinant, Replication/*genetics, Subunits},
pubstate = {published},
tppubtype = {article}
}
Przykorska A., Solecka K., Olszak K., Keith G., Nawrot B., Kuligowska E.
Wheat (Triticum vulgare) chloroplast nuclease ChSI exhibits 5' flap structure-specific endonuclease activity Journal Article
In: Biochemistry, vol. 43, no. 35, pp. 11283-94, 2004, (0006-2960 Journal Article).
Abstract | BibTeX | Tags: &, Acid, Catalysis, Chloroplasts/*enzymology, Conformation, Desorption-Ionization, DNA, Endonucleases/*chemistry/isolation, Exonucleases/chemistry/metabolism, Flap, Gov't, Hydrolysis, KEITH, Kinetics, Laser, Mass, Matrix-Assisted, Non-U.S., Nucleic, Oligonucleotides/chemical, Plant/chemistry/metabolism, purification/*metabolism, Relationship, Single-Stranded/chemistry/metabolism, Specificity, Spectrometry, Structure-Activity, Substrate, Support, synthesis/metabolism, Thermodynamics, Triticum/*enzymology
@article{,
title = {Wheat (Triticum vulgare) chloroplast nuclease ChSI exhibits 5' flap structure-specific endonuclease activity},
author = { A. Przykorska and K. Solecka and K. Olszak and G. Keith and B. Nawrot and E. Kuligowska},
year = {2004},
date = {2004-01-01},
journal = {Biochemistry},
volume = {43},
number = {35},
pages = {11283-94},
abstract = {The structure-specific ChSI nuclease from wheat (Triticum vulgare) chloroplast stroma has been previously purified and characterized in our laboratory. It is a single-strand-specific DNA and RNA endonuclease. Although the enzyme has been initially characterized and used as a structural probe, its biological function is still unknown. Localization of the ChSI enzyme inside chloroplasts, possessing their own DNA that is generally highly exposed to UV light and often affected by numerous redox reactions and electron transfer processes, might suggest, however, that this enzyme could be involved in DNA repair. The repair of some types of DNA damage has been shown to proceed through branched DNA intermediates which are substrates for the structure-specific DNA endonucleases. Thus we tested the substrate specificity of ChSI endonuclease toward various branched DNAs containing 5' flap, 5' pseudoflap, 3' pseudoflap, or single-stranded bulged structural motifs. It appears that ChSI has a high 5' flap structure-specific endonucleolytic activity. The catalytic efficiency (k(cat)/K(M)) of the enzyme is significantly higher for the 5' flap substrate than for single-stranded DNA. The ChSI 5' flap activity was inhibited by high concentrations of Mg(2+), Mn(2+), Zn(2+), or Ca(2+). However, low concentrations of divalent cations could restore the loss of ChSI activity as a consequence of EDTA pretreatment. In contrast to other known 5' flap nucleases, the chloroplast enzyme ChSI does not possess any 5'-->3' exonuclease activity on double-stranded DNA. Therefore, we conclude that ChSI is a 5' flap structure-specific endonuclease with nucleolytic activity toward single-stranded substrates.},
note = {0006-2960
Journal Article},
keywords = {&, Acid, Catalysis, Chloroplasts/*enzymology, Conformation, Desorption-Ionization, DNA, Endonucleases/*chemistry/isolation, Exonucleases/chemistry/metabolism, Flap, Gov't, Hydrolysis, KEITH, Kinetics, Laser, Mass, Matrix-Assisted, Non-U.S., Nucleic, Oligonucleotides/chemical, Plant/chemistry/metabolism, purification/*metabolism, Relationship, Single-Stranded/chemistry/metabolism, Specificity, Spectrometry, Structure-Activity, Substrate, Support, synthesis/metabolism, Thermodynamics, Triticum/*enzymology},
pubstate = {published},
tppubtype = {article}
}
Marco Valeria De, Stier Gunter, Blandin Stephanie A, de Marco Ario
The solubility and stability of recombinant proteins are increased by their fusion to NusA Journal Article
In: Biochem. Biophys. Res. Commun., vol. 322, no. 3, pp. 766–771, 2004, ISSN: 0006-291X.
Abstract | Links | BibTeX | Tags: blandin, Drug Stability, Escherichia coli Proteins, Genetic Vectors, Glutathione Transferase, Kinetics, M3i, Oxidation-Reduction, Peptide Elongation Factors, Recombinant Fusion Proteins, Recombinant Proteins, Solubility, Transcription Factors, Transcriptional Elongation Factors
@article{de_marco_solubility_2004,
title = {The solubility and stability of recombinant proteins are increased by their fusion to NusA},
author = {Valeria De Marco and Gunter Stier and Stephanie A Blandin and Ario de Marco},
doi = {10.1016/j.bbrc.2004.07.189},
issn = {0006-291X},
year = {2004},
date = {2004-01-01},
journal = {Biochem. Biophys. Res. Commun.},
volume = {322},
number = {3},
pages = {766--771},
abstract = {The new bacterial vector pETM60 enables the expression of His-tagged recombinant proteins fused to the C-terminus of NusA through a TEV protease recognition sequence. Three sequences coding for two protein domains (Xklp3A and Tep3Ag) and one membrane-bound viral protein (E8R) could not be expressed in a soluble form in bacteria. Their GST-fusions were mostly soluble but quickly degraded during purification. The same sequences cloned in pETM60 were efficiently purified by metal affinity and recovered soluble after the removal of the fusion partner. The NusA-fused constructs enabled to yield 13-20mg of fusion protein per litre of culture and 2.5-5mg of pure protein per litre of culture. Structural analysis indicated that the purified proteins were monodispersed and correctly folded. NusA has been used to raise antibodies that have been successfully used for Western blot and immunoprecipitation of NusA fusion proteins.},
keywords = {blandin, Drug Stability, Escherichia coli Proteins, Genetic Vectors, Glutathione Transferase, Kinetics, M3i, Oxidation-Reduction, Peptide Elongation Factors, Recombinant Fusion Proteins, Recombinant Proteins, Solubility, Transcription Factors, Transcriptional Elongation Factors},
pubstate = {published},
tppubtype = {article}
}
Boutabout M., Wilhelm M., Wilhelm F. X.
DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1 Journal Article
In: Nucleic Acids Res, vol. 29, no. 11, pp. 2217-22, 2001, (1362-4962 Journal Article).
Abstract | BibTeX | Tags: cerevisiae/*genetics/metabolism, DNA, Fungal/genetics, Fungal/genetics/*metabolism, Genetic, Gov't, Kinetics, Non-U.S., Nucleotides/genetics/metabolism, Polymerase/*metabolism, Retroelements/*genetics, RNA, RNA-Directed, Saccharomyces, Support, Templates
@article{,
title = {DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1},
author = { M. Boutabout and M. Wilhelm and F. X. Wilhelm},
year = {2001},
date = {2001-01-01},
journal = {Nucleic Acids Res},
volume = {29},
number = {11},
pages = {2217-22},
abstract = {The fidelity of the yeast retrotransposon Ty1 reverse transcriptase (RT) was determined by an assay based on gel electrophoresis. Steady-state kinetics analyses of deoxyribonucleotide (dNTP) incorporation at a defined primer-template site indicate that Ty1 RT misincorporates dNTP at a frequency of 0.45 x 10(-5) for the A(t):A mispair in which dATP is misincorporated opposite a template A to 6.27 x 10(-5) for the C(t):A mispair. The G(t):G and T(t):T mispairs are formed with very low efficiency. The fidelity parameters of Ty1 RT do not depend on whether RNA or DNA are copied. Relative to lentiviral RTs (HIV-1, HIV-2 or EIAV) Ty1 RT is approximately 10-fold less error prone. Our data also show that the Ty1 RT is able to recapitulate two error-generating mechanisms: extension of mismatches and non-templated addition of nucleotides at the end of a blunt-end primer-template.},
note = {1362-4962
Journal Article},
keywords = {cerevisiae/*genetics/metabolism, DNA, Fungal/genetics, Fungal/genetics/*metabolism, Genetic, Gov't, Kinetics, Non-U.S., Nucleotides/genetics/metabolism, Polymerase/*metabolism, Retroelements/*genetics, RNA, RNA-Directed, Saccharomyces, Support, Templates},
pubstate = {published},
tppubtype = {article}
}
Carnicelli D., Brigotti M., Rizzi S., Keith G., Montanaro L., Sperti S.
Nucleotides U28-A42 and A37 in unmodified yeast tRNA(Trp) as negative identity elements for bovine tryptophanyl-tRNA synthetase Journal Article
In: FEBS Lett, vol. 492, no. 3, pp. 238-41, 2001, (0014-5793 Journal Article).
Abstract | BibTeX | Tags: Acid, Adenine/chemistry, Animals, Base, Cattle, cerevisiae/genetics, Conformation, Data, Fungal/genetics/metabolism, Gov't, Kinetics, Ligase/*metabolism, Molecular, Non-U.S., Nucleic, RNA, Saccharomyces, Sequence, Species, Specificity, Substrate, Support, Transfer, Trp/chemistry/*metabolism, Tryptophan-tRNA, Uridine/chemistry
@article{,
title = {Nucleotides U28-A42 and A37 in unmodified yeast tRNA(Trp) as negative identity elements for bovine tryptophanyl-tRNA synthetase},
author = { D. Carnicelli and M. Brigotti and S. Rizzi and G. Keith and L. Montanaro and S. Sperti},
year = {2001},
date = {2001-01-01},
journal = {FEBS Lett},
volume = {492},
number = {3},
pages = {238-41},
abstract = {Wild-type bovine and yeast tRNA(Trp) are efficiently aminoacylated by tryptophanyl-tRNA synthetase both from beef and from yeast. Upon loss of modified bases in the synthetic transcripts, mammalian tRNA(Trp) retains the double recognition by the two synthetases, while yeast tRNA(Trp) loses its substrate properties for the bovine enzyme and is recognised only by the cognate synthetase. By testing chimeric bovine-yeast transcripts with tryptophanyl-tRNA synthetase purified from beef pancreas, the nucleotides responsible for the loss of charging of the synthetic yeast transcript have been localised in the anticodon arm. A complete loss of charging akin to that observed with the yeast transcript requires substitution in the bovine backbone of G37 in the anticodon loop with yeast A37 and of C28-G42 in the anticodon stem with yeast U28-A42. Since A37 does not prevent aminoacylation of the wild-type yeast tRNA(Trp) by the beef enzyme, a negative combination apparently emerges in the synthetic transcript after unmasking of U28 by loss of pseudourydilation.},
note = {0014-5793
Journal Article},
keywords = {Acid, Adenine/chemistry, Animals, Base, Cattle, cerevisiae/genetics, Conformation, Data, Fungal/genetics/metabolism, Gov't, Kinetics, Ligase/*metabolism, Molecular, Non-U.S., Nucleic, RNA, Saccharomyces, Sequence, Species, Specificity, Substrate, Support, Transfer, Trp/chemistry/*metabolism, Tryptophan-tRNA, Uridine/chemistry},
pubstate = {published},
tppubtype = {article}
}
Adamkewicz J I, Mueller C G, Hansen K E, Prud'homme W A, Thorner J
Purification and enzymic properties of Mot1 ATPase, a regulator of basal transcription in the yeast Saccharomyces cerevisiae Journal Article
In: The Journal of Biological Chemistry, vol. 275, no. 28, pp. 21158–21168, 2000, ISSN: 0021-9258.
Abstract | Links | BibTeX | Tags: Adenosine Triphosphatases, Base Sequence, Chromatography, DNA Helicases, DNA-Binding Proteins, Fungal, Gel, Gene Expression Regulation, Genetic, Kinetics, Molecular Sequence Data, Molecular Weight, Osmolar Concentration, Recombinant Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, TATA Box, TATA-Binding Protein Associated Factors, TATA-Box Binding Protein, Team-Mueller, Transcription, Transcription Factors
@article{adamkewicz_purification_2000,
title = {Purification and enzymic properties of Mot1 ATPase, a regulator of basal transcription in the yeast Saccharomyces cerevisiae},
author = {J I Adamkewicz and C G Mueller and K E Hansen and W A Prud'homme and J Thorner},
doi = {10.1074/jbc.M002639200},
issn = {0021-9258},
year = {2000},
date = {2000-07-01},
journal = {The Journal of Biological Chemistry},
volume = {275},
number = {28},
pages = {21158--21168},
abstract = {The 1867-residue Mot1 protein is a member of a superfamily of ATPases, some of which are helicases, that interact with protein-nucleic acid assemblies. Mot1 is an essential regulator of RNA polymerase II-dependent transcription in vivo and dissociates TATA box-binding protein (TBP)-DNA complexes in vitro. Mot1-(His)(6) was purified to apparent homogeneity from yeast extracts. The preparation efficiently dissociated TBP.TATA complexes, suggesting that no other protein or cofactor is required. Mot1 behaved as a non-globular monomer in hydrodynamic studies, and no association was detected between differentially tagged co-expressed Mot1 constructs. ATPase activity was stimulated about 10-fold by high ionic strength or alkaline pH, or by deletion of the N-terminal TBP-binding segment, suggesting that the N-terminal domain negatively regulates the C-terminal ATPase domain (Mot1C). Correspondingly, at moderate salt concentration, Mot1 ATPase (but not Mot1C) was stimulated textgreater/=10-fold by yeast TBP, suggesting that interaction with TBP relieves a conformational constraint in Mot1. Double- or single-stranded TATA-containing DNA did not affect ATPase activity of Mot1 or Mot1C, with or without TBP. Mot1 did not exhibit detectable helicase activity in strand displacement assays using substrates with flush ends or 5'- or 3'-overhangs. Mot1-catalyzed dissociation of TBP from DNA was not prevented by a psoralen cross-link positioned immediately preceding the TATA sequence. Thus, Mot1 most likely promotes release of TBP from TATA-containing DNA by causing a structural change in TBP itself, rather than by strand unwinding.},
keywords = {Adenosine Triphosphatases, Base Sequence, Chromatography, DNA Helicases, DNA-Binding Proteins, Fungal, Gel, Gene Expression Regulation, Genetic, Kinetics, Molecular Sequence Data, Molecular Weight, Osmolar Concentration, Recombinant Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, TATA Box, TATA-Binding Protein Associated Factors, TATA-Box Binding Protein, Team-Mueller, Transcription, Transcription Factors},
pubstate = {published},
tppubtype = {article}
}
Wilhelm M., Boutabout M., Wilhelm F. X.
Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities Journal Article
In: Biochem J, vol. 348, no. Pt 2, pp. 337-42, 2000, (0264-6021 Journal Article).
Abstract | BibTeX | Tags: &, Acid, affinity, Alignment, Amino, Calf, cerevisiae/*enzymology/*genetics, Chromatography, Cloning, Codon, coli, Comparative, Data, DNA, DNA/metabolism, Escherichia, Frames, Fusion, Genetic, Gov't, H, Heteroduplexes/metabolism, HIV-1, Homology, Integrases/chemistry/metabolism, Kinetics, Molecular, Non-U.S., Nucleic, Open, Polymerase/chemistry/isolation, Proteins/chemistry/isolation, purification/*metabolism, purification/metabolism, Reading, Recombinant, Retroelements/*genetics, Reverse, Ribonuclease, RNA-Directed, RNA/metabolism, Saccharomyces, Sequence, Study, Support, Templates, Terminator, Thymus/isolation, Transcriptase/chemistry
@article{,
title = {Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities},
author = { M. Wilhelm and M. Boutabout and F. X. Wilhelm},
year = {2000},
date = {2000-01-01},
journal = {Biochem J},
volume = {348},
number = {Pt 2},
pages = {337-42},
abstract = {Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.},
note = {0264-6021
Journal Article},
keywords = {&, Acid, affinity, Alignment, Amino, Calf, cerevisiae/*enzymology/*genetics, Chromatography, Cloning, Codon, coli, Comparative, Data, DNA, DNA/metabolism, Escherichia, Frames, Fusion, Genetic, Gov't, H, Heteroduplexes/metabolism, HIV-1, Homology, Integrases/chemistry/metabolism, Kinetics, Molecular, Non-U.S., Nucleic, Open, Polymerase/chemistry/isolation, Proteins/chemistry/isolation, purification/*metabolism, purification/metabolism, Reading, Recombinant, Retroelements/*genetics, Reverse, Ribonuclease, RNA-Directed, RNA/metabolism, Saccharomyces, Sequence, Study, Support, Templates, Terminator, Thymus/isolation, Transcriptase/chemistry},
pubstate = {published},
tppubtype = {article}
}
Schröter H, Mueller C G, Meese K, Nordheim A
Synergism in ternary complex formation between the dimeric glycoprotein p67SRF, polypeptide p62TCF and the c-fos serum response element Journal Article
In: The EMBO journal, vol. 9, no. 4, pp. 1123–1130, 1990, ISSN: 0261-4189.
Abstract | BibTeX | Tags: Base Sequence, Chloroquine, Gene Expression Regulation, Genetic, Glycosylation, HeLa Cells, Humans, Kinetics, Macromolecular Substances, Molecular Sequence Data, Nuclear Proteins, Oligonucleotide Probes, Plasmids, Polymerase Chain Reaction, Protein-Tyrosine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-fos, Proto-Oncogenes, Serum Response Factor, Team-Mueller, Transcription, Transcription Factors
@article{schroter_synergism_1990,
title = {Synergism in ternary complex formation between the dimeric glycoprotein p67SRF, polypeptide p62TCF and the c-fos serum response element},
author = {H Schröter and C G Mueller and K Meese and A Nordheim},
issn = {0261-4189},
year = {1990},
date = {1990-04-01},
journal = {The EMBO journal},
volume = {9},
number = {4},
pages = {1123--1130},
abstract = {Transcriptional regulation of the c-fos proto-oncogene requires the serum response element (SRE) which is complexed by a multi-protein assembly observed both in vitro and in vivo. Two protein factors, p67SRF and p62TCF (previously called p62), are required to interact with the SRE for efficient induction of c-fos by serum. By quantitative band shift electrophoresis we measure at least a 50-fold increase in SRE affinity for p67SRF/p62TCF over p67SRF alone. Stoichiometrically we determine that the ternary complex with p62TCF involves p67SRF in dimeric form. We demonstrate that p67SRF is a glycosylated nuclear transcription factor carrying terminal N-acetylglucosamine (GlcNAc) as a post-translational modification. A proteolytic limit digestion product, approximately 13 kd in size, was generated from the p67SRF-SRE complex. This p67SRF-core domain binds SRE, can dimerize with p67SRF and is still able to form a ternary complex with p62TCF. Therefore, three functional activities can be ascribed to this small p67SRF-core domain: specific DNA binding, dimerization and interaction with p62TCF. We demonstrate that these functions map within the p67SRF core fragment containing the region between amino acids 93 and 222.},
keywords = {Base Sequence, Chloroquine, Gene Expression Regulation, Genetic, Glycosylation, HeLa Cells, Humans, Kinetics, Macromolecular Substances, Molecular Sequence Data, Nuclear Proteins, Oligonucleotide Probes, Plasmids, Polymerase Chain Reaction, Protein-Tyrosine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-fos, Proto-Oncogenes, Serum Response Factor, Team-Mueller, Transcription, Transcription Factors},
pubstate = {published},
tppubtype = {article}
}
Kappler Christine, Kabbouh M, Hetru Charles, Durst F, Hoffmann Jules A
Characterization of three hydroxylases involved in the final steps of biosynthesis of the steroid hormone ecdysone in Locusta migratoria (Insecta, Orthoptera) Journal Article
In: J. Steroid Biochem., vol. 31, no. 6, pp. 891–898, 1988, ISSN: 0022-4731.
Abstract | BibTeX | Tags: Animals, Biological, Ecdysone, Grasshoppers, hoffmann, Kinetics, M3i, Mixed Function Oxygenases, Models, NAD, NADP, Subcellular Fractions
@article{kappler_characterization_1988,
title = {Characterization of three hydroxylases involved in the final steps of biosynthesis of the steroid hormone ecdysone in Locusta migratoria (Insecta, Orthoptera)},
author = {Christine Kappler and M Kabbouh and Charles Hetru and F Durst and Jules A Hoffmann},
issn = {0022-4731},
year = {1988},
date = {1988-12-01},
journal = {J. Steroid Biochem.},
volume = {31},
number = {6},
pages = {891--898},
abstract = {It is most generally accepted that the last three enzymatic reactions in the biosynthetic pathway of ecdysone are, in this order, the hydroxylations at positions C-25, C-22 and C-2. Using high specific activity tritiated ecdysone precursors (2,22,25-trideoxyecdysone, 2,22-dideoxyecdysone and 2-deoxyecdysone) we have characterized the hydroxylases involved in these reactions, in the major biosynthetic tissue of ecdysone, i.e. the prothoracic glands. We show that C-2 hydroxylase is a mitochondrial oxygenase which differs from conventional cytochrome P-450-dependent monooxygenases by its relative insensitivity to CO. In contrast, C-22 and C-25 hydroxylases appear as classical cytochrome P-450 monooxygenases; C-22 hydroxylase is a mitochondrial enzyme whereas our data point to a microsomal localization of the C-25 hydroxylase.},
keywords = {Animals, Biological, Ecdysone, Grasshoppers, hoffmann, Kinetics, M3i, Mixed Function Oxygenases, Models, NAD, NADP, Subcellular Fractions},
pubstate = {published},
tppubtype = {article}
}
Goltzené F, Lagueux Marie, Charlet Maurice, Hoffmann Jules A
The follicle cell epithelium of maturing ovaries of Locusta migratoria: a new biosynthetic tissue for ecdysone Journal Article
In: Hoppe-Seyler's Z. Physiol. Chem., vol. 359, no. 10, pp. 1427–1434, 1978, ISSN: 0018-4888.
Abstract | BibTeX | Tags: Animals, Ecdysone, Epithelium, Female, Grasshoppers, hoffmann, Kinetics, M3i, Oocytes, Ovarian Follicle, Ovary, Sexual Maturation
@article{goltzene_follicle_1978,
title = {The follicle cell epithelium of maturing ovaries of Locusta migratoria: a new biosynthetic tissue for ecdysone},
author = {F Goltzené and Marie Lagueux and Maurice Charlet and Jules A Hoffmann},
issn = {0018-4888},
year = {1978},
date = {1978-10-01},
journal = {Hoppe-Seyler's Z. Physiol. Chem.},
volume = {359},
number = {10},
pages = {1427--1434},
abstract = {Follicle cells of maturing ovaries of Locusta migratoria are demonstrated to synthesize the moulting hormone ecdysone (2beta,3beta,14alpha,22R,25-pentahydroxy-5beta-cholest-7-en-6-one). Studies of secretory kinetics under in vitro conditions show that the intensity of hormone secretion is strictly dependent on the stage of maturation of the excised ovaries.},
keywords = {Animals, Ecdysone, Epithelium, Female, Grasshoppers, hoffmann, Kinetics, M3i, Oocytes, Ovarian Follicle, Ovary, Sexual Maturation},
pubstate = {published},
tppubtype = {article}
}
Koolman J, Hoffmann Jules A, Karlson P
Sulphage esters as inactivation products of ecdysone in Locusta migratoria Journal Article
In: Hoppe-Seyler's Z. Physiol. Chem., vol. 354, no. 9, pp. 1043–1048, 1973, ISSN: 0018-4888.
BibTeX | Tags: Animals, Biological, Cattle, Chromatography, Ecdysone, Electrophoresis, Esterases, Glucosidases, Glucuronidase, Grasshoppers, hoffmann, Hydrogen-Ion Concentration, Hydrolysis, Ion Exchange, Isotope Labeling, Kinetics, Larva, Liver, M3i, Metamorphosis, Paper, Plants, Saccharomyces cerevisiae, Snails, Sulfatases, Sulfur Radioisotopes, Sulfuric Acids, Swine, Thin Layer, Time Factors, Tritium
@article{koolman_sulphage_1973,
title = {Sulphage esters as inactivation products of ecdysone in Locusta migratoria},
author = {J Koolman and Jules A Hoffmann and P Karlson},
issn = {0018-4888},
year = {1973},
date = {1973-09-01},
journal = {Hoppe-Seyler's Z. Physiol. Chem.},
volume = {354},
number = {9},
pages = {1043--1048},
keywords = {Animals, Biological, Cattle, Chromatography, Ecdysone, Electrophoresis, Esterases, Glucosidases, Glucuronidase, Grasshoppers, hoffmann, Hydrogen-Ion Concentration, Hydrolysis, Ion Exchange, Isotope Labeling, Kinetics, Larva, Liver, M3i, Metamorphosis, Paper, Plants, Saccharomyces cerevisiae, Snails, Sulfatases, Sulfur Radioisotopes, Sulfuric Acids, Swine, Thin Layer, Time Factors, Tritium},
pubstate = {published},
tppubtype = {article}
}