Calpain Purification Through Calpastatin and Calcium: Strategy and Procedures.

TitleCalpain Purification Through Calpastatin and Calcium: Strategy and Procedures.
Publication TypeJournal Article
Year of Publication2019
AuthorsNguyen, H. Huy, P. Tompa, and K. Pauwels
JournalMethods Mol Biol
Volume1929
Pagination233-244
Date Published2019
ISSN1940-6029
KeywordsBiotinylation, Calcium, Calcium-Binding Proteins, Calpain, Chromatography, Affinity, Escherichia coli, Humans
Abstract

This chapter describes the strategy and procedures for the calcium-mediated affinity purification of calpain. The affinity capture method exploits the reversible binding properties of calpain's intrinsically disordered protein (IDP) inhibitor, calpastatin. IDPs are easily produced in heterologous expression hosts and purified to homogeneity. Combining these properties with in vivo biotinylation leads to a simplified purification strategy whereby biotinylated human calpastatin domain 1 (hCSD1) can capture calpain efficiently from a complex biological mixture with only a single chromatographic step and in a considerably reduced time. Our approach is generally applicable through the in vivo biotinylation of any IDP of interest in order to capture its binding partner in a calcium- and chelator-based protocol.

DOI10.1007/978-1-4939-9030-6_15
Alternate JournalMethods Mol. Biol.
PubMed ID30710277
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