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Degron-controlled protein degradation in Escherichia coli: New Approaches and Parameters

Protein degron tags have proven uniquely useful for characterization of gene function. Degrons mediate quick depletion, usually within minutes, of a protein of interest – allowing researchers to characterize cellular responses to the loss of function. To develop a general purpose degron tool in E. c...

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Autores principales: Cronan, Glen E., Kuzminov, Andrei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10659297/
https://www.ncbi.nlm.nih.gov/pubmed/37986802
http://dx.doi.org/10.1101/2023.11.08.566101
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author Cronan, Glen E.
Kuzminov, Andrei
author_facet Cronan, Glen E.
Kuzminov, Andrei
author_sort Cronan, Glen E.
collection PubMed
description Protein degron tags have proven uniquely useful for characterization of gene function. Degrons mediate quick depletion, usually within minutes, of a protein of interest – allowing researchers to characterize cellular responses to the loss of function. To develop a general purpose degron tool in E. coli, we sought to build upon a previously characterized system of SspB-dependent inducible protein degradation. For this, we created a family of expression vectors containing a destabilized allele of SspB, capable of a rapid and nearly perfect “off-to-on” induction response. Using this system, we demonstrated control over several enzymes of DNA metabolism, but also found with other substates apparent limitations of a SspB-dependent system. Several degron target proteins were degraded too slowly to affect their complete depletion during active growth, whereas others appeared completely refractory to degron-promoted degradation. We demonstrated that a model substrate, beta-galactosidase, was positively recognized as a degron substrate, but failed to be degraded by the ClpXP protease — demonstrating an apparently unknown mechanism of protease resistance. Thus, only a minority of our, admittedly biased, selection of degron substates proved amenable to rapid SspB-catalyzed degradation. We conclude that substrate-dependence of the SspB system remains a critical factor for the success of this degron system. For substrates that prove degradable, we provide a series of titratable SspB-expression vehicles.
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spelling pubmed-106592972023-11-20 Degron-controlled protein degradation in Escherichia coli: New Approaches and Parameters Cronan, Glen E. Kuzminov, Andrei bioRxiv Article Protein degron tags have proven uniquely useful for characterization of gene function. Degrons mediate quick depletion, usually within minutes, of a protein of interest – allowing researchers to characterize cellular responses to the loss of function. To develop a general purpose degron tool in E. coli, we sought to build upon a previously characterized system of SspB-dependent inducible protein degradation. For this, we created a family of expression vectors containing a destabilized allele of SspB, capable of a rapid and nearly perfect “off-to-on” induction response. Using this system, we demonstrated control over several enzymes of DNA metabolism, but also found with other substates apparent limitations of a SspB-dependent system. Several degron target proteins were degraded too slowly to affect their complete depletion during active growth, whereas others appeared completely refractory to degron-promoted degradation. We demonstrated that a model substrate, beta-galactosidase, was positively recognized as a degron substrate, but failed to be degraded by the ClpXP protease — demonstrating an apparently unknown mechanism of protease resistance. Thus, only a minority of our, admittedly biased, selection of degron substates proved amenable to rapid SspB-catalyzed degradation. We conclude that substrate-dependence of the SspB system remains a critical factor for the success of this degron system. For substrates that prove degradable, we provide a series of titratable SspB-expression vehicles. Cold Spring Harbor Laboratory 2023-11-09 /pmc/articles/PMC10659297/ /pubmed/37986802 http://dx.doi.org/10.1101/2023.11.08.566101 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
spellingShingle Article
Cronan, Glen E.
Kuzminov, Andrei
Degron-controlled protein degradation in Escherichia coli: New Approaches and Parameters
title Degron-controlled protein degradation in Escherichia coli: New Approaches and Parameters
title_full Degron-controlled protein degradation in Escherichia coli: New Approaches and Parameters
title_fullStr Degron-controlled protein degradation in Escherichia coli: New Approaches and Parameters
title_full_unstemmed Degron-controlled protein degradation in Escherichia coli: New Approaches and Parameters
title_short Degron-controlled protein degradation in Escherichia coli: New Approaches and Parameters
title_sort degron-controlled protein degradation in escherichia coli: new approaches and parameters
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10659297/
https://www.ncbi.nlm.nih.gov/pubmed/37986802
http://dx.doi.org/10.1101/2023.11.08.566101
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