Purification of recombinant ESCRT-III proteins and their use in atomic force microscopy and In vitro binding and phosphorylation assays

Luisa Capalbo; Ioanna Mela; Maria Alba Abad; A. Arockia Jeyaprakash; J. Michael Edwardson; Pier Paolo D’Avino

doi:10.1007/978-1-4939-9492-2_15

Purification of recombinant ESCRT-III proteins and their use in atomic force microscopy and In vitro binding and phosphorylation assays

Luisa Capalbo, Ioanna Mela, Maria Alba Abad, A. Arockia Jeyaprakash, J. Michael Edwardson, Pier Paolo D’Avino

School of Biological Sciences

Research output: Chapter in Book/Report/Conference proceeding › Chapter

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.

Original language	English
Title of host publication	Methods in Molecular Biology
Publisher	HUMANA PRESS INC
Pages	203-217
Number of pages	15
DOIs	https://doi.org/10.1007/978-1-4939-9492-2_15
Publication status	E-pub ahead of print - 28 Jun 2019

Publication series

Name	Methods in Molecular Biology
Volume	1998
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Keywords

atomic force microscopy
CHMP4C
kinase assay
protein binding
protein purification

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10.1007/978-1-4939-9492-2_15

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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 Methods in Molecular Biology (pp. 203-217). (Methods in Molecular Biology; Vol. 1998). HUMANA PRESS INC. https://doi.org/10.1007/978-1-4939-9492-2_15

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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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author = "Luisa Capalbo and Ioanna Mela and Abad, {Maria Alba} and Jeyaprakash, {A. Arockia} and Edwardson, {J. Michael} and D{\textquoteright}Avino, {Pier Paolo}",

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Capalbo, L, Mela, I, Abad, MA, Jeyaprakash, AA, Edwardson, JM & D’Avino, PP 2019, Purification of recombinant ESCRT-III proteins and their use in atomic force microscopy and In vitro binding and phosphorylation assays. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1998, HUMANA PRESS INC, pp. 203-217. https://doi.org/10.1007/978-1-4939-9492-2_15

Purification of recombinant ESCRT-III proteins and their use in atomic force microscopy and In vitro binding and phosphorylation assays. / Capalbo, Luisa; Mela, Ioanna; Abad, Maria Alba; Jeyaprakash, A. Arockia; Edwardson, J. Michael; D’Avino, Pier Paolo.

Methods in Molecular Biology. HUMANA PRESS INC, 2019. p. 203-217 (Methods in Molecular Biology; Vol. 1998).

Research output: Chapter in Book/Report/Conference proceeding › Chapter

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