RECOMBINANT CALPAIN-II - IMPROVED EXPRESSION SYSTEMS AND PRODUCTION OF A 105A ACTIVE-SITE MUTANT FOR CRYSTALLOGRAPHY

The bacterial production of recombinant rat calpain II has been improv ed greatly by the use of two compatible plasmids for the two subunits. The calpain small subunit C-terminal fragment (21 kDa) was expressed from a new A15-based vector created by cloning T7 control elements int o pACYC177, This vector is compatible with the ColE1-based pET-24d(+) vector containing the calpain large subunit, and the yield of calpain activity was increased at least 16-fold by co-expression from these tw o vectors, A high level of activity was also obtained from a bicistron ic construct containing both subunit cDNAs under the control of one T7 promoter. The addition of a C-terminal His-tag to the large subunit s implified purification without affecting subunit association or enzyme activity. The active-site cysteine 105 was mutated to alanine, causin g complete loss of activity, The yield of purified C105A-calpain II (8 0 + 21 kDa) dimer following three column chromatography steps was 10 m g/l of cell culture, This provides a purified calpain, stable to autol ysis and oxidation, which is likely to facilitate crystallization in b oth the presence and absence of calcium.