RT Journal Article
SR Electronic
T1 A uniform benchmark for testing ssrA-derived degrons in the Escherichia coli ClpXP pathway
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.08.26.457770
DO 10.1101/2021.08.26.457770
A1 Klimecka, Maria Magdalena
A1 Antosiewicz, Anna
A1 Izert, Matylda Anna
A1 Szybowska, Patrycja Emanuela
A1 Twardowski, Piotr Krzysztof
A1 Delaunay, Clara
A1 Górna, Maria Wiktoria
YR 2021
UL http://biorxiv.org/content/early/2021/09/22/2021.08.26.457770.abstract
AB 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.