1. Development of molecular methods for the comprehensive evaluation of stress conditions affecting forest trees : dissertationClaudia D'Ercole, 2025, doctoral dissertation Keywords: protein complexes, adhirons, nanobodies, plant stress, immunoreagents, protoplasts, ascorbate peroxidase, dissertations
Published in RUNG: 04.02.2025; Views: 402; Downloads: 1
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3. Computational study of the HLTF ATPase remodeling domain suggests its activity on dsDNA and implications in damage toleranceMartin Ljubič, Claudia D'Ercole, Yossma Waheed, Ario De Marco, Jure Borišek, Matteo De March, 2024, original scientific article Keywords: DNA repairing mechanism, protein modeling, chromatin remodeler, helicase-like transcription factor
Published in RUNG: 15.11.2024; Views: 751; Downloads: 2
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4. Native agarose gels and contact blotting as means to optimize the protocols for the formation of antigen-ligand complexesClaudia D'Ercole, Ario De Marco, 2024, published scientific conference contribution abstract Abstract: Background. Protein complexes provide valuable biological information but can be difficult to handle [1]. Therefore, technical advancements designed to improve their manipulation are al-ways useful.
Methods. We investigated the opportunity to exploit native agarose gels and the contact bot method for the transfer of native proteins to membranes as means for optimizing the conditions for obtaining stable complexes [2]. As a simple model of protein-protein interactions, an anti-gen-ligand complex was used in which both proteins were fused to reporters.
Results. At each step, it was possible to visualize both the antigen, fused to a fluorescent pro-tein, and the ligand, fused to a monomeric ascorbate peroxidase (APEX) and, as such, a way to tune the protocol. The conditions for the complex formation were adapted by modifying the buffer conditions, the concentration of the proteins and of the cross-linkers.
Conclusions. The procedure is rapid, inexpensive, and the several detection opportunities al-lows for both the monitoring of complex stability and the preservation of the functionality of its components, which is critical for understanding their biomedical implications and supporting drug discovery. The overall protocol represents a handy alternative to gel filtration, uses very standard and ubiquitous equipment, and can be implemented rapidly and without specific train-ing.
References:
[1] O. Puig, F. Caspary, G. Rigaut, B. Rutz, E. Bouveret, E. Bragado-Nilsson, M. Wilm, B. Séraphin. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods. (San Diego, Calif.), 24(3), 218–229
[2] C. Sakuma, M. Nakagawa, Y. Tomioka, T. Maruyama, K. Entzminger, J.K. Fleming, T. Shibata, Y. Kurosawa, C.J. Okumura, T. Arakawa, T. Akuta. Western blotting of native proteins from agarose gels. Biotechniques. 2022 May;72(5):207-218
Keywords: protein complexes, contact blotting, native agarose gels, protein interaction
Published in RUNG: 17.06.2024; Views: 1332; Downloads: 1
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5. Instruct-ERIC network : biophysical characterization of antigen-nanobody complexesClaudia D'Ercole, 2024, published scientific conference contribution abstract Abstract: Forest environments are exposed to multiple stressful factors of both abiotic and biotic nature which may lead to their massive decline [1]. Understanding the molecular mechanisms of specific stress conditions and monitoring the fluctuations of reliable forest plant biomarkers with affordable methods would be instrumental for assessing stress levels over the time. Ascorbate peroxidase (APX) represents a suitable plant biomarker. APX is a hydrogen peroxide-scavenging enzyme the critical role of which has been described in several plants, both herbaceous and woody. Its activity generally increases under oxidative stress during which its peroxide detoxifying function is part of the wider ascorbate-glutathione cycle [2]. The development of reagents to detect such fluctuations would help the evaluation of plant physiological conditions. In this study, nanobodies (Nbs) targeting APX have been identified. Nbs correspond to the variable domain of heavy chain-only antibodies derived from camelids. They are small (15 kDa), stable, and can be easily produced in bacteria fused to different protein tags according to the downstream applications [3]. After their isolation by biopanning against soluble APX, they have been produced and underwent a biophysical characterization in combination with their antigen (APX-Nb complex) to identify the best binders in terms of stability and affinity. The protein complex characterization was supported by Instruct-ERIC and mainly performed at the BIOCEV institute of Prague. Data from Mass Photometry and Dynamic Light scattering evidenced the formation of the protein complexes, whereas the preliminary data of Hydrogen-Deuterium Exchange Mass Spectrometry, performed with the aim of identifying the residues involved in the paratope/epitope interface, were insufficient to clarify the issue and rather suggested that the interaction has low affinity. This indication was then confirmed by ELISA assay. The combination of multiple methods allowed a comprehensive sample characterization which will require further structural analyses to provide a complete picture of the APX-Nb complex.
[1] G. Marie. B. C. M. B. C. Walters, “Forest decline and tree mortality in a southeastern Ohio oak-hickory forest,” Ohio Journal of Science , vol. 97, 1997.
[2] O. Chew, J. Whelan, and A. H. Millar, “Molecular Definition of the Ascorbate-Glutathione Cycle in Arabidopsis Mitochondria Reveals Dual Targeting of Antioxidant Defenses in Plants,” Journal of Biological Chemistry, vol. 278, no. 47, 2003, doi: 10.1074/jbc.M307525200.
[3] S. Muyldermans, “A guide to: generation and design of nanobodies,” FEBS J, vol. 288, no. 7, pp. 2084–2102, Apr. 2021, doi: 10.1111/febs.15515.
Keywords: nanobody, ascorbate peroxidase, plant stress, protein complex, biophysical methodologies
Published in RUNG: 31.05.2024; Views: 1471; Downloads: 0
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6. A practical guide for the quality evaluation of fluobodies/chromobodiesUrša Štrancar, Claudia D'Ercole, Lucia Cikatricisová, Mirna Nakić, Matteo De March, Ario De Marco, 2024, original scientific article Keywords: fluorescent proteins, protein stability, protein degradation
Published in RUNG: 16.05.2024; Views: 1496; Downloads: 7
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7. Biological applications of synthetic binders isolated from a conceptually new adhiron libraryClaudia D'Ercole, Matteo De March, Gianluca Veggiani, Sandra Oloketuyi, Rossella Svigelj, Ario De Marco, 2023, original scientific article Keywords: synthetic library, adhirons, affimers, C-reactive protein, SpyCatcher
Published in RUNG: 17.10.2023; Views: 2127; Downloads: 5
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10. Macroporous epoxy-based monoliths functionalized with anti-CD63 nanobodies for effective isolation of extracellular vesicles in urineJulia Neumair, Claudia D'Ercole, Matteo De March, Martin Elsner, Michael Seidel, Ario De Marco, 2023, original scientific article Keywords: extracellular vesicles, monolith chromatography, nanobodies, functionalitazion strategies
Published in RUNG: 27.03.2023; Views: 2793; Downloads: 14
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