Abstract
Some proteins represent members of conserved families, meaning that their domain structure can be easily predicted by comparison to homologous proteins whose structures have been solved experimentally. Many other proteins, however, do not share significant detectable homology with other proteins, often as results of high amounts of coiled-coil structure and/or intrinsically unstructured regions. These proteins include many whose aggregation is linked to human disease.
Here we present a refined and reliable workflow for identifying the domains of such proteins, through cloning of multiple alternative fragments, and testing whether they form soluble, folded structures when expressed as recombinant peptides in E. coli, through the use of size exclusion chromatography. By using Gateway recombination for cloning, these fragments can then be rapidly transferred to alternate vectors for testing in mammalian cells. We then specifically illustrate its use for proteins that form pathological aggregates in disease, mapping not just their basic domain structures but also the specific subdomains responsible for aggregation.
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Acknowledgments
We thank our colleagues, past and present, in the Bradshaw (Rijeka), Korth (Düsseldorf), Hart (Grenoble), Walkinshaw, and Millar (Edinburgh) groups for their discussion and ideas over time regarding these methods.
This work was funded by the Croatian Science Foundation (Hrvatska zaklada za znanost, IP-2018-01-9424 and DOK-2018-09-5395). Equipment used in this work was supplied by the European Regional Development Fund (RC.2.2.06.0001) and grants from the Alexander von Humboldt Foundation and University of Rijeka (1157).
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Zaharija, B., Bradshaw, N.J. (2023). Mapping the Domain Structure and Aggregation Propensity of Proteins Using a Gateway Plasmid Vector System. In: Cieplak, A.S. (eds) Protein Aggregation. Methods in Molecular Biology, vol 2551. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2597-2_39
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DOI: https://doi.org/10.1007/978-1-0716-2597-2_39
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