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Calpain Purification Through Calpastatin and Calcium: Strategy and Procedures

  • Hung Huy Nguyen
  • Peter Tompa
  • Kris PauwelsEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)

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.

Key words

Intrinsically disordered proteins IDPs Affinity chromatography Calpain-2 Human m-calpain In vivo biotinylation AviTag Chemical biotinylation BirA co-expression Affinity capture 

Notes

Acknowledgments

The authors thank Ivo Van Overstraeten for excellent technical assistance. This work was supported in part by a pilot grant of Stichting Alzheimer Onderzoek (P#12017), and K.P. is the recipient of a FWO long-term postdoctoral fellowship (#1218713).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Hung Huy Nguyen
    • 1
    • 2
  • Peter Tompa
    • 1
    • 2
    • 3
  • Kris Pauwels
    • 1
    • 2
    Email author
  1. 1.VIB-VUB Centre for Structural Biology (VIB CSB), Vlaams Instituut voor Biotechnologie (VIB)BrusselsBelgium
  2. 2.Structural Biology Brussels (SBB), Department of Biotechnology (DBIT)Vrije Universiteit Brussel (VUB)BrusselsBelgium
  3. 3.Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of SciencesBudapestHungary

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