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Calpain pp 13-27 | Cite as

Bacterial Expression and Purification of Calpains

  • Christian-Scott E. McCartney
  • Peter L. DaviesEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1915)

Abstract

The production of recombinant proteins has been a cornerstone of the study of protein structure and function. As an example, the expression and purification of recombinant rat calpain-2 in bacteria was essential for solving the first crystal structures of the calpains in both calcium-free and calcium-bound forms. Here we describe the production and purification of recombinant rat calpain-2 from Escherichia coli using anion-exchange, affinity, and size-exclusion chromatographies. The heterodimeric enzyme is produced from a stable two-plasmid system. The order in which the protocol is carried out has been optimized to reduce unnecessary concentration and dialysis steps. The typical yield of this multi-domain enzyme from 4 L of E. coli culture is about 20 mg. The production of whole structures for the other calpain family members has been fraught with difficulty. To circumvent this roadblock, a certain amount of structure-function information can be gleaned about these other calpain isoforms by expressing just their protease core. These “mini-calpains” have been useful for X-ray co-crystallography with calpain inhibitors.

Here we also present a variation of the whole enzyme production and purification protocol optimized for the expression and purification of the calpain-1 and calpain-3 protease cores (mini-calpains).

Key words

Calpain-2 Calpain-1 Calpain-3 Protease core Protein purification Recombinant protein Protein expression Affinity chromatography Size-exclusion chromatography Anion-exchange chromatography 

Notes

Acknowledgments

We thank Dr. Laurie Graham for her suggestion for monitoring the success of sonication using the Bradford assay. This work was funded by the Canadian Institute of Health Research. PLD holds a Canada Research Chair in Protein Engineering.

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Copyright information

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

Authors and Affiliations

  • Christian-Scott E. McCartney
    • 1
  • Peter L. Davies
    • 1
    Email author
  1. 1.Department of Biomedical and Molecular SciencesQueen’s UniversityKingstonCanada

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