Production of Purified Proteins Tagged with ssrA-Derived Degrons and Their Degradation by the Escherichia coli ClpXP...

The ssrA degron is widely employed in fusion proteins to regulate protein stability in bacteria or to serve as an interaction module. These uses take advantage of the ssrA tag's dual functionality-its ability to bind the SspB adaptor and to recruit the ClpXP protease. A comprehensive comparison of these functions across a standardized set of degron variants can be helpful in the development of variants specifically optimized for particular applications and their use in targeted protein degradation systems. Here, we describe a simple and efficient purification protocol for a set of ssrA-based degrons and a degradation assay that can be used as the primary assay to compare the effectiveness of degradation of degron-tagged proteins.