On-off switching of enzymatic reaction by recombinant calmodulin on a solid-phase matrix.
A fusion protein consisting of human calmodulin (CaM) and glutathione S-transferase (GST) was produced by gene fusion. The fusion protein was overexpressed in Escherichia coli as a soluble form and purified with one-step affinity chromatography using glutathione-Sepharose. The protein had the modulating activity of CaM and the binding capability to glutathione of GST. Phosphodiesterase, which is a CaM dependent enzyme, was activated by the fusion protein, with the Ca2+ level equal to the level equivalent to a native CaM. Furthermore, CaM could be immobilized on a solid-phase matrix through the use of GST moiety while its modulating activity was retained. Phosphodiesterase activity was switched on and off by the immobilized CaM with or without Ca2+, and repeated use of CaM was demonstrated.