1. 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: 148; Downloads: 0 This document has many files! More... |
2. 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: 227; Downloads: 2 Full text (667,85 KB) This document has many files! More... |
3. Novel bispecific nanobody mitigates experimental intestinal inflammation in mice by targeting TNF-▫$\alpha$▫ and IL-23p19 bioactivitiesJiewen Wang, Guangbo Kang, Huiying Lu, Ario De Marco, Haibin Yuan, Zelin Feng, Mengxue Gao, Xiaoli Wang, Huahong Wang, Xiaolan Zhang, 2024, original scientific article Keywords: bispecific constructs, nanobodies, intestinal inflammation Published in RUNG: 27.03.2024; Views: 479; Downloads: 3 Full text (12,21 MB) This document has many files! More... |
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8. A concise guide to choosing suitable gene expression systems for recombinant protein productionAnja Schütz, Frank Bernhard, Nick Berrow, Johannes F. Buyel, Frederico Ferreira-da-Silva, Jurgen Haustraete, Joop Van den Heuvel, Jan-Erik Hoffmann, Ario De Marco, Yoav Peleg, 2023, original scientific article Keywords: recombinant protein, protein expression options, post-translational modifications Published in RUNG: 06.11.2023; Views: 805; Downloads: 5 Full text (3,14 MB) This document has many files! More... |
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10. 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: 887; Downloads: 3 Full text (2,23 MB) This document has many files! More... |