Abstract
The physicochemical characterization of protein aggregates yields an important contribution to further our understanding on many diseases for which the formation of protein aggregates is one of the pathological hallmarks. On the other hand, bacterial inclusion bodies (IBs) have recently been shown to be highly pure proteinaceous aggregates of a few hundred nanometers, produced by recombinant bacteria supporting the biological activities of the embedded polypeptides. Despite the wide spectrum of uses of IBs as functional and biocompatible materials upon convenient engineering, very few is known about their physicochemical properties.
In this chapter we present methods for the characterization of protein aggregates as particulate materials relevant to their physicochemical and nanoscale properties.
Specifically, we describe the use of dynamic light scattering (DLS) for sizing, nanoparticle tracking analysis for sizing and counting, and zeta potential measurements for the determination of colloidal stability. To study the morphology of protein aggregates we present the use of atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cryo-transmission electron microscopy (cryo-TEM) will be used for the determination of the internal structuration. Moreover, wettability and nanomechanical characterization can be performed using contact angle (CA) and force spectroscopic AFM (FS-AFM) measurements of the proteinaceous nanoparticles, respectively. Finally, the 4′4-dithiodipyridine (DTDP) method is presented as a way of relatively quantifying accessible sulfhydryl groups in the structure of the nanoparticle .
The physical principles of the methods are briefly described and examples are given to help clarify capabilities of each technique.
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Acknowledgments
The authors are grateful for the financial support received from MOTHER and Mol4Bio (MAT2016-80826-R and PID2019- 105622RBI00) granted by the DGI (Spain), GenCat (SGR-918 and SGR-229) financed by DGR (Catalunya), the SpanishMinistry of Economy and Competitiveness (MINECO) through the “Severo Ochoardquo; Programme for Centres of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S), the COST Action CA15126 Between Atom and Cell, Fundació La Marató de TV3 (Nr. 201812). This study has been also supported by the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), an initiative funded by the VI National R&D&I Plan, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. The NTA, DLS and zeta potential measurements have been performed by the Biomaterial Processing and Nanostructuring Unit (U6) of the ICTS “NANBIOSISrdquo;, a unit of the CIBER network in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) located at the Institute of Materials Science of Barcelona (ICMAB-CSIC). J.G. is also grateful to MINECO for a “Ramon y Cajalrdquo; fellowship (Nr. RYC-2017-22614), the Max Planck Society through the Max Planck Partner Group “Dynamic Biomimetics for Cancer Immunotherapyrdquo; in collaboration with the Max Planck Institute for Medical Research (Heidelberg, Germany).
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Martínez-Miguel, M. et al. (2022). Methods for the Characterization of Protein Aggregates. In: Garcia Fruitós, E., Arís Giralt, A. (eds) Insoluble Proteins. Methods in Molecular Biology, vol 2406. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1859-2_29
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DOI: https://doi.org/10.1007/978-1-0716-1859-2_29
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