A Uniform Benchmark for Testing SsrA-Derived Degrons in the Escherichia coli ClpXP Degradation Pathway

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 activiti...

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Published in:	Molecules (Basel, Switzerland) Vol. 26; no. 19; p. 5936
Main Authors:	Klimecka, Maria Magdalena, Antosiewicz, Anna, Izert, Matylda Anna, Szybowska, Patrycja Emanuela, Twardowski, Piotr Krzysztof, Delaunay, Clara, Górna, Maria Wiktoria
Format:	Journal Article
Language:	English
Published:	Switzerland MDPI AG 30-09-2021 MDPI
Subjects:	Adenosine triphosphatase Adenosine Triphosphate - metabolism Amino acids Bacteria Benchmarking Benchmarks Binding Biodegradation Carrier Proteins - genetics Carrier Proteins - metabolism Chromatography ClpXP degradation degron E coli Efficiency Endopeptidase Clp - genetics Endopeptidase Clp - metabolism Escherichia coli - genetics Escherichia coli - metabolism Escherichia coli Proteins - genetics Escherichia coli Proteins - metabolism Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism In vivo methods and tests Models, Molecular protease Protein Binding Proteins SspB ssrA Substrate Specificity Workflow protease ssrA degradation SspB ClpXP degron
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Summary:	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 the development 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1420-3049 1420-3049
DOI:	10.3390/molecules26195936