TY - JOUR T1 - A uniform benchmark for testing ssrA-derived degrons in the Escherichia coli ClpXP pathway JF - bioRxiv DO - 10.1101/2021.08.26.457770 SP - 2021.08.26.457770 AU - Maria Magdalena Klimecka AU - Anna Antosiewicz AU - Matylda Anna Izert AU - Patrycja Emanuela Szybowska AU - Piotr Krzysztof Twardowski AU - Clara Delaunay AU - Maria Wiktoria Górna Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/09/22/2021.08.26.457770.abstract N2 - The ssrA degron is commonly used in fusion proteins to control protein stability in bacteria or as an interaction module. These applications often rely on the modular activities of the ssrA tag in binding to the SspB adaptor and in engaging the ClpXP protease. However, a comparison of these activities for a substantial standard set of degron variants has not been conducted previously, which may hinder developments of new variants optimized exclusively for one application. Here, we strive to establish a benchmark that will facilitate the comparison of ssrA variants under uniform conditions. In our workflow, we included methods for expression and purification of ClpX, ClpP, SspB and eGFP-degrons, assays of ClpX ATPase activity, of eGFP-degron binding to SspB and for measuring eGFP-degron degradation in vitro and in vivo. Using uniform, precise and sensitive methods under the same conditions on a range of eGFP-degrons allowed us to determine subtle differences in their properties that can affect their potential applications. Our findings can serve as a reference and a resource for developing targeted protein degradation approaches.SUMMARY This work lays standards for assays used to compare engagement of SspB and ClpXP by a set of ssrA-derived degrons that can be used to fine-tune tools for protein stability control. ER -