Expression and characterization of the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II using the baculovirus expression system.

Sf9 cells infected with the recombinant mouse CaMKII-alpha (Ca2+/calmodulin dependent kinase II) baculovirus expressed 12-15 mg of MCaMKII-alpha per liter of cells. Approximately 50% of the MCaMKII-alpha activity could be purified using a CaM-Sepharose affinity column. The purified MCaMKII-alpha had a M(rapp) of 50 kDa by SDS-PAGE and a native Mr of 600 kDa. MCaMKII-alpha, like rat brain CaMKII, had an A0.5 for CaM of 100 nM, a Km for syntide-2 of 8 microM, and was able to generate Ca2(+)-independent activity by autophosphorylation. The baculovirus system expressed large quantities of MCaMKII-alpha with characteristics similar to the rat brain CaMKII, thus providing an expression system for the detailed biochemical analysis of MCaMKII-alpha.