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

Research output: Chapter in Book/Report/Conference proceedingChapter

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 languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHUMANA PRESS INC
Pages203-217
Number of pages15
DOIs
Publication statusE-pub ahead of print - 28 Jun 2019

Publication series

NameMethods in Molecular Biology
Volume1998
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • atomic force microscopy
  • CHMP4C
  • kinase assay
  • protein binding
  • protein purification

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