Bacterial Expression and Purification of Calpains.

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 4L of E. coli culture is about 20mg. 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).