Abstract
The endosomal sorting complex required for transport (ESCRT)-III proteins are known to assemble into filaments that mediate membrane remodeling and fission in various biological processes, including the formation of endosomal multivesicular bodies, viral budding, cytokinesis, plasma membrane repair, nuclear pore quality control, nuclear envelope reformation, and neuron pruning. The study of the regulation and function of ESCRT-III proteins is therefore crucial to understand these events and requires a combination of in vivo and in vitro experimental techniques. Here we describe two protocols for the purification of human and Drosophila ESCRT-III proteins from bacteria and their use in in vitro phosphorylation assays and atomic force microscopy experiments on membrane lipid bilayers. These protocols can also be applied for the purification of other proteins that are insoluble when expressed in bacteria.
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Acknowledgments
This work was supported by Cancer Research UK grant C12296/A12541 and Biotechnology and Biological Research Council grant BB/R001227/1 to PPD. IM and JME were supported by Biotechnology and Biological Research Council grant BB/J018236/1. AAJ is supported by a Well come Trust Senior Research Fellowship 202811.
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Capalbo, L., Mela, I., Abad, M.A., Jeyaprakash, A.A., Edwardson, J.M., D’Avino, P.P. (2019). Purification of Recombinant ESCRT-III Proteins and Their Use in Atomic Force Microscopy and In Vitro Binding and Phosphorylation Assays. In: Culetto, E., Legouis, R. (eds) The ESCRT Complexes. Methods in Molecular Biology, vol 1998. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9492-2_15
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DOI: https://doi.org/10.1007/978-1-4939-9492-2_15
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