Biomedicals from a soil bug

Dammeyer, Thorben

doi:10.4161/bbug.3.1.17739

Cited by 2 publications

(2 citation statements)

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“…The significant differences in GC content of the YFPs result in no significant differences in expression and fluorescence levels. However, from Figure 3 it can be interpreted that the high GC construct expresses slightly lower protein levels in E.coli compared to P.putida KT2440 which is in accordance with the previously postulated high plasticity of P.putida KT2440 expressing foreign genes with different GC contents [25]. To verify the functionality of our periplasmic PelB-leader sequence expression construct variant and the novel engineered periplasmic fluorescent reporter protein, we transformed vectors pTDpelB-C_TwinStrepeYFP and pTDpelB-C_TwinStrepsfYFP to both reference organisms.…”

Section: Resultssupporting

confidence: 86%

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Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein

2013

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BackgroundIn current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains.ResultsHere, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs.ConclusionThe broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co-localization studies. In addition, the new tools facilitate metabolic engineering and yield assessment for cytoplasmic or periplasmic protein production in a number of different expression hosts when yields in one initially selected are insufficient.

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Section: Resultssupporting

confidence: 86%

“…The PelB signal sequence (ssPelB) has proven to be successful in periplasmic production and precise processing of the pre-proteins of recombinant single-chain scFv antibodies using similar vector chassis in P . putida KT2440 [13,25]. …”

Section: Resultsmentioning

confidence: 99%

Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein

2013

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Ionic control of porin permeability in bacteria

Muniz

Hagting

Evans

et al. 2022

Preprint

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Bacterial porins permit permeation of hydrophilic nutrients and antibiotics across the outer membrane but also contribute to proton leak from the periplasmic space, suggesting that their activity might be dynamically regulated. Here we show, in Escherichia coli, that porin permeability is controlled by changes in periplasmic ions, inhibited by periplasmic acidification, thereby limiting proton loss during electron transport chain activity, and enhanced during starvation, promoting nutrient uptake. Growth in glucose increases periplasmic potassium through activating the voltage-gated channel Kch, triggering enhanced porin permeation and membrane action potentials. This metabolic control of porin permeability explains the recognized decrease in antibiotic susceptibility when bacteria are grown in lipid media and the impact of mutations in central metabolism genes on drug resistance, identifying Kch as a therapeutic target to improve bacterial killing by antibiotics.

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Biomedicals from a soil bug

Cited by 2 publications

References 29 publications

Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein

Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein

Ionic control of porin permeability in bacteria

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