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 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.
VIB Center for Structural Biology (CSB), Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.|Structural Biology Brussels (SBB), Vrije Universiteit Brussel, Brussels, Belgium.
Hung Huy
HH
Nguyen
VIB Center for Structural Biology (CSB), Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.|Structural Biology Brussels (SBB), Vrije Universiteit Brussel, Brussels, Belgium.
Mihaly
M
Varadi
VIB Center for Structural Biology (CSB), Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.|Structural Biology Brussels (SBB), Vrije Universiteit Brussel, Brussels, Belgium.|Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary.
Peter
P
Tompa
VIB Center for Structural Biology (CSB), Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium.|Structural Biology Brussels (SBB), Vrije Universiteit Brussel, Brussels, Belgium.
Kris
http://orcid.org/0000-0002-7341-0890
K
Pauwels
D019921:Biotinylation; D065096:Calcium Chelating Agents / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D019295:Computational Biology; D004926:Escherichia coli; D006801:Humans; D007368:Interferometry; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D000072417:Protein Domains; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D065096:Calcium Chelating Agents; D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; C033668:calpastatin; D002154:Calpain; C569870:CAPN2 protein, human