Protein purification to analyze AAA+ proteolytic machine in vitro

Abstract

The ATP-dependent ClpXP protease of Escherichia coli consists of two subunits, the ClpP subunit, which has the proteolytic activity and the AAA motor ClpX, which mechanically unfolds and translocates substrates for ClpP degradation. In order to investigate the mechanical properties of ClpXP during substrate unfolding using magnetical tweezers, here we optimize protocols to purify ClpP and a model substrate based on human Filamin A from E. coli lysates by a combination of metal affinity chromatography and fast performance liquid chromatography (FPLC). ClpX purification was challenging and remains to be improved. Importantly, a HaloTag protein molecule was fused to the Filamin substrate, which allowed covalent bonding to surfaces or fluorescent molecules. We analyzed the mechanical properties of a single Filamin A substrate using atomic force spectroscopy (AFM) and found good correlation with previous single-molecule experiments based on optical tweezers. AFM experiments also demonstrate the successful binding of the HaloTag moiety to a modified glass surface. The results show that the study of ClpXP-mediated degradation of proteins by magnetic tweezers has the potential to unveil the mechanics of protein degradation inside cells.