Purification of Recombinant Calpastatin Expressed in Escherichia coli

Calpastatin is an endogenous inhibitor protein specific for calpain (1 ,2 ). Other calpain-inhibiting reagents such as EDTA, E-64, leupeptin, and calpain inhibitor I (N -acetyl-L-leucyl-L-leucyl-L-norleucinal), are frequently used in attempts to assess the role of calpain in degradation of target proteins in cell lysates. However, these reagents are not strictly specific for calpain (for instance, cathepsins B, H, and L are inhibited by E-64, and the proteasome is inhibited by calpain inhibitor I), so that we cannot logically establish that a given degradation is mediated by calpain even if it is inhibited by these reagents. It is clearly important to use inhibitors specific for calpains (3 ), and at present calpastatin alone provides the necessary specificity.