ON-OFF SWITCHING OF ENZYMATIC-REACTION BY RECOMBINANT CALMODULIN ON ASOLID-PHASE MATRIX

A fusion protein consisting of human calmodulin (CaM) and glutathione S-transferase (GST) was produced by gene fusion. The fusion protein wa s overexpressed in Escherichia coli as a soluble form and purified wit h one-step affinity chromatography using glutathione-Sepharose. The pr otein had the modulating activity of CaM and the binding capability to glutathione of GST. Phosphodiesterase, which is a CaM dependent enzym e, was activated by the fusion protein, with the Ca2+ level equal to t he level equivalent to a native CaM. Furthermore, CaM could be immobil ized on a solid-phase matrix through the use of GST moiety while its m odulating activity was retained. Phosphodiesterase activity was switch ed on and off by the immobilized CaM with or without Ca2+, and repeate d use of CaM was demonstrated.