Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Cronan GE

PROVIDER: S-EPMC10659297 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Degron-controlled protein degradation in <i>Escherichia coli</i>: New Approaches and Parameters.

Cronan Glen E GE Kuzminov Andrei A

bioRxiv : the preprint server for biology 20231109

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 <i>E. coli</i>, 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 all ...[more]

PMID: 37986802

Similar Datasets

Project description:Easy-to-perform, fast, and inexpensive methods of differentiation of Escherichia coli strains beyond the species level are highly required. Herein two new, original tools for genotyping of E. coli isolates are proposed. The first of the developed method, a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) test uses a highly variable fliC gene, encoding the H antigen as a molecular target. The designing of universal pair of primers and selection of the optimal restriction enzyme RsaI was preceded by in silico comparative analysis of the sequences of the genes coding for 53 different serotypes of H-antigen (E. coli flagellin). The target fragments of E. coli genomes for MLST method were selected on the basis of bioinformatics analysis of complete sequences of 16 genomes of E. coli. Initially, seven molecular targets were proposed (seven pairs of primers) and five of them were found useful for effective genotyping of E. coli strains. Both developed methods revealed high differentiation power, and a high genetic diversity of the strains tested was observed. Within the group of 71 strains tested, 29 and 47 clusters were revealed with fliC RFLP-PCR and MLST methods, respectively. Differentiation of the strains with the reference BOX-PCR method revealed 31 different genotypes. The in silico analysis revealed that the discriminatory power of the new MLST method is comparable to the Pasteur and Achtman schemes and is higher than the discriminatory power of the method developed by Clermont. From the epidemiology point of view, the outcomes of our investigation revealed that in most cases, the patients were infected with unique strains, probably from environmental sources. However, some strains isolated from different patients of the wards of pediatrics, internal medicine, and neurology were classified to the same genotype when the results of all three methods were taken into account. It could suggest that they were transferred between the patients.

Project description:Conditional protein degradation is a powerful tool for controlled protein knockdown. The auxin-inducible degron (AID) technology uses a plant auxin to induce depletion of degron-tagged proteins, and it has been shown to be functional in several non-plant eukaryotes. In this study, we demonstrated AID-based protein knockdown in an industrially important oleaginous yeast Yarrowia lipolytica. Using the mini-IAA7 (mIAA7) degron derived from Arabidopsis IAA7, coupled with an Oryza sativa TIR1 (OsTIR1) plant auxin receptor F-box protein (expressed from the copper-inducible MT2 promoter), C-terminal degron-tagged superfolder GFP could be degraded in Yarrowia lipolytica upon addition of copper and the synthetic auxin 1-Naphthaleneacetic acid (NAA). However, leaky degradation of the degron-tagged GFP in the absence of NAA was also noted. This NAA-independent degradation was largely eliminated by replacing the wild-type OsTIR1 and NAA with the OsTIR1F74A variant and the auxin derivative 5-Ad-IAA, respectively. Degradation of the degron-tagged GFP was rapid and efficient. However, Western blot analysis revealed cellular proteolytic cleavage within the mIAA7 degron sequence, leading to the production of a GFP sub-population lacking an intact degron. The utility of the mIAA7/OsTIR1F74A system was further explored in controlled degradation of a metabolic enzyme, β-carotene ketolase, which converts β-carotene to canthaxanthin via echinenone. This enzyme was tagged with the mIAA7 degron and expressed in a β-carotene producing Y. lipolytica strain that also expressed OsTIR1F74A controlled by the MT2 promoter. By adding copper and 5-Ad-IAA at the time of culture inoculation, canthaxanthin production was found to be reduced by about 50% on day five compared to the control culture without adding 5-Ad-IAA. This is the first report that demonstrates the efficacy of the AID system in Y. lipolytica. Further improvement of AID-based protein knockdown in Y. lipolytica may be achieved by preventing proteolytic removal of the mIAA7 degron tag.

Dataset Information

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

Publications

Degron-controlled protein degradation in <i>Escherichia coli</i>: New Approaches and Parameters.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets