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  1. Article ; Online: An open-source automated PEG precipitation assay to measure the relative solubility of proteins with low material requirement

    Marc Oeller / Pietro Sormanni / Michele Vendruscolo

    Scientific Reports, Vol 11, Iss 1, Pp 1-

    2021  Volume 10

    Abstract: Abstract The solubility of proteins correlates with a variety of their properties, including function, production yield, pharmacokinetics, and formulation at high concentrations. High solubility is therefore a key requirement for the development of ... ...

    Abstract Abstract The solubility of proteins correlates with a variety of their properties, including function, production yield, pharmacokinetics, and formulation at high concentrations. High solubility is therefore a key requirement for the development of protein-based reagents for applications in life sciences, biotechnology, diagnostics, and therapeutics. Accurate solubility measurements, however, remain challenging and resource intensive, which limits their throughput and hence their applicability at the early stages of development pipelines, when long-lists of candidates are typically available in minute amounts. Here, we present an automated method based on the titration of a crowding agent (polyethylene glycol, PEG) to quantitatively assess relative solubility of proteins using about 200 µg of purified material. Our results demonstrate that this method is accurate and economical in material requirement and costs of reagents, which makes it suitable for high-throughput screening. This approach is freely-shared and based on a low cost, open-source liquid-handling robot. We anticipate that this method will facilitate the assessment of the developability of proteins and make it substantially more accessible.
    Keywords Medicine ; R ; Science ; Q
    Language English
    Publishing date 2021-11-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Editorial

    Angelo Toto / Pietro Sormanni / Cristina Paissoni / Vladimir N. Uversky

    Frontiers in Molecular Biosciences, Vol

    Intrinsically Disordered Proteins and Regions: The Challenge to the Structure-Function Relationship

    2022  Volume 9

    Keywords intrinsically disordered protein ; intrinsically disordered region ; structure-function continuum ; protein-protein interaction ; multifunctional protein ; protein structure ; Biology (General) ; QH301-705.5
    Language English
    Publishing date 2022-07-01T00:00:00Z
    Publisher Frontiers Media S.A.
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Automated optimisation of solubility and conformational stability of antibodies and proteins

    Angelo Rosace / Anja Bennett / Marc Oeller / Mie M. Mortensen / Laila Sakhnini / Nikolai Lorenzen / Christian Poulsen / Pietro Sormanni

    Nature Communications, Vol 14, Iss 1, Pp 1-

    2023  Volume 15

    Abstract: Antibodies find key applications in research, diagnostics, and therapeutics, but their development can be impeded by poor stability or solubility. Here the authors developed a computational strategy that enables antibody optimisation, without affecting ... ...

    Abstract Antibodies find key applications in research, diagnostics, and therapeutics, but their development can be impeded by poor stability or solubility. Here the authors developed a computational strategy that enables antibody optimisation, without affecting functionality.
    Keywords Science ; Q
    Language English
    Publishing date 2023-04-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Rapid and accurate in silico solubility screening of a monoclonal antibody library

    Pietro Sormanni / Leanne Amery / Sofia Ekizoglou / Michele Vendruscolo / Bojana Popovic

    Scientific Reports, Vol 7, Iss 1, Pp 1-

    2017  Volume 9

    Abstract: Abstract Antibodies represent essential tools in research and diagnostics and are rapidly growing in importance as therapeutics. Commonly used methods to obtain novel antibodies typically yield several candidates capable of engaging a given target. The ... ...

    Abstract Abstract Antibodies represent essential tools in research and diagnostics and are rapidly growing in importance as therapeutics. Commonly used methods to obtain novel antibodies typically yield several candidates capable of engaging a given target. The development steps that follow, however, are usually performed with only one or few candidates since they can be resource demanding, thereby increasing the risk of failure of the overall antibody discovery program. In particular, insufficient solubility, which may lead to aggregation under typical storage conditions, often hinders the ability of a candidate antibody to be developed and manufactured. Here we show that the selection of soluble lead antibodies from an initial library screening can be greatly facilitated by a fast computational prediction of solubility that requires only the amino acid sequence as input. We quantitatively validate this approach on a panel of nine distinct monoclonal antibodies targeting nerve growth factor (NGF), for which we compare the predicted and measured solubilities finding a very close match, and we further benchmark our predictions with published experimental data on aggregation hotspots and solubility of mutational variants of one of these antibodies.
    Keywords Medicine ; R ; Science ; Q
    Language English
    Publishing date 2017-08-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article: Targeting disordered proteins with small molecules using entropy

    Heller, Gabriella T / Pietro Sormanni / Michele Vendruscolo

    Trends in biochemical sciences. 2015 Sept., v. 40

    2015  

    Abstract: The human proteome includes many disordered proteins. Although these proteins are closely linked with a range of human diseases, no clinically approved drug targets them in their monomeric forms. This situation arises, at least in part, from the current ... ...

    Abstract The human proteome includes many disordered proteins. Although these proteins are closely linked with a range of human diseases, no clinically approved drug targets them in their monomeric forms. This situation arises, at least in part, from the current lack of understanding of the mechanisms by which small molecules bind proteins that do not fold into well-defined conformations. To explore possible solutions to this problem, we discuss quite generally how an overall decrease in the free energy associated with intermolecular binding can originate from different combinations of enthalpic and entropic contributions. We then consider more specifically a mechanism of binding by which small molecules can affect the conformational space of a disordered protein by creating an entropic expansion in which more conformations of the protein become populated.
    Keywords Gibbs free energy ; drugs ; entropy ; human diseases ; humans ; proteins ; proteome
    Language English
    Dates of publication 2015-09
    Size p. 491-496.
    Publishing place Elsevier Ltd
    Document type Article
    ZDB-ID 194220-7
    ISSN 0968-0004 ; 0376-5067
    ISSN 0968-0004 ; 0376-5067
    DOI 10.1016/j.tibs.2015.07.004
    Database NAL-Catalogue (AGRICOLA)

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  6. Article ; Online: A chemical kinetic basis for measuring translation initiation and elongation rates from ribosome profiling data.

    Ajeet K Sharma / Pietro Sormanni / Nabeel Ahmed / Prajwal Ciryam / Ulrike A Friedrich / Günter Kramer / Edward P O'Brien

    PLoS Computational Biology, Vol 15, Iss 5, p e

    2019  Volume 1007070

    Abstract: Analysis methods based on simulations and optimization have been previously developed to estimate relative translation rates from next-generation sequencing data. Translation involves molecules and chemical reactions, hence bioinformatics methods ... ...

    Abstract Analysis methods based on simulations and optimization have been previously developed to estimate relative translation rates from next-generation sequencing data. Translation involves molecules and chemical reactions, hence bioinformatics methods consistent with the laws of chemistry and physics are more likely to produce accurate results. Here, we derive simple equations based on chemical kinetic principles to measure the translation-initiation rate, transcriptome-wide elongation rate, and individual codon translation rates from ribosome profiling experiments. Our methods reproduce the known rates from ribosome profiles generated from detailed simulations of translation. By applying our methods to data from S. cerevisiae and mouse embryonic stem cells, we find that the extracted rates reproduce expected correlations with various molecular properties, and we also find that mouse embryonic stem cells have a global translation speed of 5.2 AA/s, in agreement with previous reports that used other approaches. Our analysis further reveals that a codon can exhibit up to 26-fold variability in its translation rate depending upon its context within a transcript. This broad distribution means that the average translation rate of a codon is not representative of the rate at which most instances of that codon are translated, and it suggests that translational regulation might be used by cells to a greater degree than previously thought.
    Keywords Biology (General) ; QH301-705.5
    Subject code 410
    Language English
    Publishing date 2019-05-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: Identifying A- and P-site locations on ribosome-protected mRNA fragments using Integer Programming

    Nabeel Ahmed / Pietro Sormanni / Prajwal Ciryam / Michele Vendruscolo / Christopher M. Dobson / Edward P. O’Brien

    Scientific Reports, Vol 9, Iss 1, Pp 1-

    2019  Volume 14

    Abstract: Abstract Identifying the A- and P-site locations on ribosome-protected mRNA fragments from Ribo-Seq experiments is a fundamental step in the quantitative analysis of transcriptome-wide translation properties at the codon level. Many analyses of Ribo-Seq ... ...

    Abstract Abstract Identifying the A- and P-site locations on ribosome-protected mRNA fragments from Ribo-Seq experiments is a fundamental step in the quantitative analysis of transcriptome-wide translation properties at the codon level. Many analyses of Ribo-Seq data have utilized heuristic approaches applied to a narrow range of fragment sizes to identify the A-site. In this study, we use Integer Programming to identify the A-site by maximizing an objective function that reflects the fact that the ribosome’s A-site on ribosome-protected fragments must reside between the second and stop codons of an mRNA. This identifies the A-site location as a function of the fragment’s size and its 5′ end reading frame in Ribo-Seq data generated from S. cerevisiae and mouse embryonic stem cells. The correctness of the identified A-site locations is demonstrated by showing that this method, as compared to others, yields the largest ribosome density at established stalling sites. By providing greater accuracy and utilization of a wider range of fragment sizes, our approach increases the signal-to-noise ratio of underlying biological signals associated with translation elongation at the codon length scale.
    Keywords Medicine ; R ; Science ; Q
    Language English
    Publishing date 2019-04-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  8. Article ; Online: Supersaturated proteins are enriched at synapses and underlie cell and tissue vulnerability in Alzheimer's disease

    Rosie Freer / Pietro Sormanni / Prajwal Ciryam / Burkhard Rammner / Silvio O. Rizzoli / Christopher M. Dobson / Michele Vendruscolo

    Heliyon, Vol 5, Iss 11, Pp e02589- (2019)

    2019  

    Abstract: Neurodegenerative disorders progress across the brain in characteristic spatio-temporal patterns. A better understanding of the factors underlying the specific cell and tissue vulnerability responsible for such patterns could help identify the molecular ... ...

    Abstract Neurodegenerative disorders progress across the brain in characteristic spatio-temporal patterns. A better understanding of the factors underlying the specific cell and tissue vulnerability responsible for such patterns could help identify the molecular origins of these conditions. To investigate these factors, based on the observation that neurodegenerative disorders are closely associated with the presence of aberrant protein deposits, we made the hypothesis that the vulnerability of cells and tissues is associated to the overall levels of supersaturated proteins, which are those most metastable against aggregation. By analyzing single-cell transcriptomic and subcellular proteomics data on healthy brains of ages much younger than those typical of disease onset, we found that the most supersaturated proteins are enriched in cells and tissues that succumb first to neurodegeneration. Then, by focusing the analysis on a metastable subproteome specific to Alzheimer's disease, we show that it is possible to recapitulate the pattern of disease progression using data from healthy brains. We found that this metastable subproteome is significantly enriched for synaptic processes and mitochondrial energy metabolism, thus rendering the synaptic environment dangerous for aggregation. The present identification of protein supersaturation as a signature of cell and tissue vulnerability in neurodegenerative disorders could facilitate the search for effective treatments by providing clearer points of intervention.
    Keywords Biophysics ; Neuroscience ; Alzheimer's disease ; Protein supersaturation ; Neuronal vulnerability ; Protein homeostasis ; Science (General) ; Q1-390 ; Social sciences (General) ; H1-99
    Subject code 612
    Language English
    Publishing date 2019-11-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  9. Article ; Online: Delivery of Native Proteins into C. elegans Using a Transduction Protocol Based on Lipid Vesicles

    Michele Perni / Francesco A. Aprile / Sam Casford / Benedetta Mannini / Pietro Sormanni / Christopher M. Dobson / Michele Vendruscolo

    Scientific Reports, Vol 7, Iss 1, Pp 1-

    2017  Volume 8

    Abstract: Abstract The nematode worm Caenorhabditis elegans (C. elegans) is a versatile and widely used animal model for in vivo studies of a broad range of human diseases, in particular for understanding their genetic origins and for screening drug candidates. ... ...

    Abstract Abstract The nematode worm Caenorhabditis elegans (C. elegans) is a versatile and widely used animal model for in vivo studies of a broad range of human diseases, in particular for understanding their genetic origins and for screening drug candidates. Nevertheless, the challenges associated with the administration of native proteins to C. elegans have limited the range of applications of this animal model in protein-based drug discovery programs. Here, we describe a readily usable protocol for the transduction of native proteins in C. elegans, which is based on the encapsulation of the proteins of interest within cationic lipid vesicles, prior to their administration to worms. This procedure limits the degradation of the proteins in the guts of the animals, and promotes their adsorption into body tissues. To illustrate the efficacy of this approach we apply it to deliver an antibody designed to inhibit α-synuclein aggregation, and show that it can lead to the rescue of the disease phenotype in a C. elegans model of Parkinson’s disease. As this transduction protocol is fast and inexpensive, we anticipate that it will be readily applicable to protein-based drug discovery studies that utilize C. elegans as a model organism.
    Keywords Medicine ; R ; Science ; Q
    Subject code 500
    Language English
    Publishing date 2017-11-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  10. Article ; Online: A method of predicting the in vitro fibril formation propensity of Aβ40 mutants based on their inclusion body levels in E. coli

    Kalyani Sanagavarapu / Elisabeth Nüske / Irem Nasir / Georg Meisl / Jasper N. Immink / Pietro Sormanni / Michele Vendruscolo / Tuomas P. J. Knowles / Anders Malmendal / Celia Cabaleiro-Lago / Sara Linse

    Scientific Reports, Vol 9, Iss 1, Pp 1-

    2019  Volume 14

    Abstract: Abstract Overexpression of recombinant proteins in bacteria may lead to their aggregation and deposition in inclusion bodies. Since the conformational properties of proteins in inclusion bodies exhibit many of the characteristics typical of amyloid ... ...

    Abstract Abstract Overexpression of recombinant proteins in bacteria may lead to their aggregation and deposition in inclusion bodies. Since the conformational properties of proteins in inclusion bodies exhibit many of the characteristics typical of amyloid fibrils. Based on these findings, we hypothesize that the rate at which proteins form amyloid fibrils may be predicted from their propensity to form inclusion bodies. To establish a method based on this concept, we first measured by SDS-PAGE and confocal microscopy the level of inclusion bodies in E. coli cells overexpressing the 40-residue amyloid-beta peptide, Aβ40, wild-type and 24 charge mutants. We then compared these results with a number of existing computational aggregation propensity predictors as well as the rates of aggregation measured in vitro for selected mutants. Our results show a strong correlation between the level of inclusion body formation and aggregation propensity, thus demonstrating the power of this approach and its value in identifying factors modulating aggregation kinetics.
    Keywords Medicine ; R ; Science ; Q
    Language English
    Publishing date 2019-03-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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