In vivo biotinylated calpastatin improves the affinity purification of human m-calpain

Nguyen, Hung Huy and Volkov, Alexander N. and Vandenbussche, Guy and Tompa, Péter and Pauwels, Kris (2018) In vivo biotinylated calpastatin improves the affinity purification of human m-calpain. PROTEIN EXPRESSION AND PURIFICATION, 145. pp. 77-84. ISSN 1046-5928


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Recently we established a novel affinity purification method for calpain by exploiting the specific and reversible binding properties of its intrinsically disordered protein inhibitor, calpastatin. The immobilization strategy relied on the strength and specificity of the biotin - streptavidin interaction. Here, we report an improved and optimized method that even enables the general applicability of in vivo biotinylated (intrinsically disordered) proteins in any affinity capture strategy. Since in vitro chemical biotinylation is only accomplished with reagents that lack exact site specificity, it can not only cause sample heterogeneity but it can also hamper the functionality of the biotinylated molecules. Therefore, we have developed a recombinant expression protocol to produce in vivo biotinylated human calpastatin domain 1 (hCSD1) in Escherichia coli. We have experimentally verified that the biotinylated polypeptide tag is compatible with the intrinsically disordered state of hCSD1 and that it does not influence the functional properties of this intrinsically disordered protein (IDP). The in vivo biotinylated hCSD1 was then used without the need of any prepurification step prior to the affinity capturing of its substrate, human m-calpain. This leads to a simplified purification strategy that allows capturing the calpain efficiently from a complex biological mixture with only a single chromatogaphic step and in a considerably reduced timeframe. Our approach is generally applicable through the in vivo biotinylation of any IDP of interest, and its practical implementation will showcase the power to exploit the properties of IDPs in affinity capture strategies. © 2018 The Authors

Item Type: Article
Uncontrolled Keywords: Protein co-expression; IDP; Calpain-2; BirA; Biotin ligase; BAP-tag; AviTag; affinity chromatography
Subjects: Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia
Depositing User: MTMT SWORD
Date Deposited: 20 Mar 2018 07:26
Last Modified: 20 Mar 2018 08:29

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