A protocol for preparing nucleotide-free KaiC monomer

The hexameric form of the KaiC protein is a core of the cyanobacterial biological clock, and its enzymatic activities exhibit circadian periodicity. The instability of the monomeric form of nucleotide-free KaiC has precluded its storage and detailed analyses of the activities of the reassembled hexamer. Here, we provide a protocol for preparing nucleotide-free KaiC monomer that is stable in solution and for triggering its reassembly into intact KaiC hexamer by the addition of ATP. A phosphate buffer containing glutamic acid and arginine enhanced the stability of KaiC monomer considerably. In addition, we found that reassembled KaiC hexamer was functionally active as the intact hexamer. This protocol provides a methodological basis for further analyses of first-turnover events of the ATPase/autokinase/autophosphatase activities of the KaiC hexamer.