Bacterial Expression and Purification of Calpains.

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).