Calpain Purification Through Calpastatin and Calcium: Strategy and Procedures
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 wordsIntrinsically disordered proteins IDPs Affinity chromatography Calpain-2 Human m-calpain In vivo biotinylation AviTag Chemical biotinylation BirA co-expression Affinity capture
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).
- 14.Thompson VF, Goll DE (2000) Purification of μ-calpain, m-calpain, and calpastatin from animal tissues. In: Methods in molecular biology. Humana Press, Totowa, NJ, pp 3–16Google Scholar