Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin.
Calpains are calcium-activated proteases that have biomedical and biotechnological potential. Their activity is tightly regulated by their endogenous inhibitor, calpastatin that binds to the enzyme only in the presence of calcium. Conventional approaches to purify calpain comprise multiple chromatog...
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doaj-4cb9228e4fdc4aea92c85f8c77c866452020-11-25T01:46:00ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-01123e017412510.1371/journal.pone.0174125Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin.Hung Huy NguyenMihaly VaradiPeter TompaKris PauwelsCalpains are calcium-activated proteases that have biomedical and biotechnological potential. Their activity is tightly regulated by their endogenous inhibitor, calpastatin that binds to the enzyme only in the presence of calcium. Conventional approaches to purify calpain comprise multiple chromatographic steps, and are labor-intensive, leading to low yields. Here we report a new purification procedure for the human m-calpain based on its reversible calcium-mediated interaction with the intrinsically disordered calpastatin. We exploit the specific binding properties of human calpastatin domain 1 (hCSD1) to physically capture human m-calpain from a complex biological mixture. The dissociation of the complex is mediated by chelating calcium, upon which heterodimeric calpain elutes while hCSD1 remains immobilized onto the stationary phase. This novel affinity-based purification was compared to the conventional multistep purification strategy and we find that it is robust, it yields a homogeneous preparation, it can be scaled up easily and it rests on a non-disruptive step that maintains close to physiological conditions that allow further biophysical and functional studies.http://europepmc.org/articles/PMC5358782?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Hung Huy Nguyen Mihaly Varadi Peter Tompa Kris Pauwels |
spellingShingle |
Hung Huy Nguyen Mihaly Varadi Peter Tompa Kris Pauwels Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin. PLoS ONE |
author_facet |
Hung Huy Nguyen Mihaly Varadi Peter Tompa Kris Pauwels |
author_sort |
Hung Huy Nguyen |
title |
Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin. |
title_short |
Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin. |
title_full |
Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin. |
title_fullStr |
Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin. |
title_full_unstemmed |
Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin. |
title_sort |
affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2017-01-01 |
description |
Calpains are calcium-activated proteases that have biomedical and biotechnological potential. Their activity is tightly regulated by their endogenous inhibitor, calpastatin that binds to the enzyme only in the presence of calcium. Conventional approaches to purify calpain comprise multiple chromatographic steps, and are labor-intensive, leading to low yields. Here we report a new purification procedure for the human m-calpain based on its reversible calcium-mediated interaction with the intrinsically disordered calpastatin. We exploit the specific binding properties of human calpastatin domain 1 (hCSD1) to physically capture human m-calpain from a complex biological mixture. The dissociation of the complex is mediated by chelating calcium, upon which heterodimeric calpain elutes while hCSD1 remains immobilized onto the stationary phase. This novel affinity-based purification was compared to the conventional multistep purification strategy and we find that it is robust, it yields a homogeneous preparation, it can be scaled up easily and it rests on a non-disruptive step that maintains close to physiological conditions that allow further biophysical and functional studies. |
url |
http://europepmc.org/articles/PMC5358782?pdf=render |
work_keys_str_mv |
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