BIOLOGICALLY-ACTIVE MONOMERIC AND HETERODIMERIC RECOMBINANT HUMAN CALPAIN-I PRODUCED USING THE BACULOVIRUS EXPRESSION SYSTEM

Calpain I is a heterodimeric protein that is part of a family of calci um-activated intracellular cysteine proteases presumed to play a role in mediating signals transduced by calcium. Expression of bioactive re combinant human calpain I has been achieved using the baculovirus expr ession system, by either co-infection with two viruses, each expressin g one of the subunits, or infection with a single virus containing bot h subunits. The similar to 80 kDa catalytic subunit exhibited calcium- dependent proteolytic activity when expressed alone or with the simila r to 30 kDa regulatory subunit. Baculoviral recombinant calpain I appe ared fully active in that the catalytic subunit in unpurified cell ext racts exhibited calcium-dependent autocatalytic cleavage at the correc t locus. The amount of similar to 80 kDa subunit accumulated at steady state was greatly increased by co-expression of the similar to 30 kDa subunit, suggesting a possible role for enzyme stabilization by the l atter subunit. The recombinant human calpain I was purified to near ho mogeneity and compared with purified native human erythrocyte calpain I. The recombinant and native enzymes had equivalent inhibition consta nts for structurally diverse calpain inhibitors, identical calcium act ivation profiles, and similar specific activities, demonstrating the s uitability of using the recombinant protein for studies of the native enzyme.