[23] Endopeptidase Clp: ATP-dependent Clp protease from Escherichia coli

Publisher Summary Escherichia coli Clp protease is a multicomponent protease that has an adenosine triphosphate (ATP)-activated proteolytic activity and an ATPase activity that is activated by proteins and peptides. This chapter describes purification and properties of two components of Clp protease—namely, ClpP and ClpA. These two components by themselves form an active complex, referred as “ClpAP protease,” responsible for degradation of specific classes of proteins. The regulatory subunit of Clp protease, ClpA, can be overexpressed in mostly soluble form in E. coli cells, both under its own promoter and under strong promoters such as p L and p tac on multicopy plasmids. Repeated freezing and thawing of purified ClpA and relatively short exposures to temperatures above 10° lead to losses of activity. ATP and nonhydrolyzable analogs of ATP stabilize ClpA. ClpA and ClpP form a tight complex in the presence of MgCl 2 and ATP or the nonhydrolyzable analog, ATP γ S. The ClpAP complex is composed of a dodecamer of ClpP and a hexamer of ClpA.