Controlling and quantifying protein concentration in <i>Escherichia coli</i>

Speer, Shannon L.; Guseman, Alex J.; Patteson, Jon B.; Ehrmann, Brandie M.; Pielak, Gary J.

doi:10.1002/pro.3637

Cited by 11 publications

(20 citation statements)

References 30 publications

(33 reference statements)

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“…The E. coli cytosol contains up to 40% proteins w/v and has a pH between 7.4 and 7.6 under normal growth conditions [26,27]. When overexpressed, nontoxic small proteins commonly achieve an intracellular concentration above 2 mM and account for about 30% of the total protein content [28]. Bacterial overexpression of NT thus captures several features of the native environment of the silk gland.…”

Section: Nt Adopts a Tight Structure Inside Cellsmentioning

confidence: 99%

High intracellular stability of the spidroin N‐terminal domain in spite of abundant amyloidogenic segments revealed by in‐cell hydrogen/deuterium exchange mass spectrometry

et al. 2019

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Proteins require an optimal balance of conformational flexibility and stability in their native environment to ensure their biological functions. A striking example is spidroins, spider silk proteins, which are stored at extremely high concentrations in soluble form, yet undergo amyloid‐like aggregation during spinning. Here, we elucidate the stability of the highly soluble N‐terminal domain (NT) of major ampullate spidroin 1 in the Escherichia coli cytosol as well as in inclusion bodies containing fibrillar aggregates. Surprisingly, we find that NT, despite being largely composed of amyloidogenic sequences, showed no signs of concentration‐dependent aggregation. Using a novel intracellular hydrogen/deuterium exchange mass spectrometry (HDX‐MS) approach, we reveal that NT adopts a tight fold in the E. coli cytosol and in this manner conceals its aggregation‐prone regions by maintaining a tight fold under crowded conditions. Fusion of NT to the unstructured amyloid‐forming Aβ 40 peptide, on the other hand, results in the formation of fibrillar aggregates. However, HDX‐MS indicates that the NT domain is only partially incorporated into these aggregates in vivo . We conclude that NT is able to control its aggregation to remain functional under the extreme conditions in the spider silk gland.

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Section: Nt Adopts a Tight Structure Inside Cellsmentioning

confidence: 99%

High intracellular stability of the spidroin N‐terminal domain in spite of abundant amyloidogenic segments revealed by in‐cell hydrogen/deuterium exchange mass spectrometry

et al. 2019

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“… 2 Alternatively, the cells themselves could be used to create the calibration curves for specific strains: by measuring the number of cells in a known dilution in the flow cytometer, it would be possible to estimate the number of cells in a dilution series of the same sample measured in the plate reader. In addition, determination of the MEFL per individual GFP protein, for example by flow cytometry and liquid chromatography–mass spectrometry, 27 would be useful to gain quantitative information on how many proteins per cells are producing that fluorescence. This would be highly beneficial to enable the coupling of computational models to experimental data.…”

Section: Discussionmentioning

confidence: 99%

FlopR: An Open Source Software Package for Calibration and Normalization of Plate Reader and Flow Cytometry Data

et al. 2020

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The measurement of gene expression using fluorescence markers has been a cornerstone of synthetic biology for the past two decades. However, the use of arbitrary units has limited the usefulness of these data for many quantitative purposes. Calibration of fluorescence measurements from flow cytometry and plate reader spectrophotometry has been implemented previously, but the tools are disjointed. Here we pull together, and in some cases improve, extant methods into a single software tool, written as a package in the R statistical framework. The workflow is validated using Escherichia coli engineered to express green fluorescent protein (GFP) from a set of commonly used constitutive promoters. We then demonstrate the package’s power by identifying the time evolution of distinct subpopulations of bacteria from bulk plate reader data, a task previously reliant on laborious flow cytometry or colony counting experiments. Along with standardized parts and experimental methods, the development and dissemination of usable tools for quantitative measurement and data analysis will benefit the synthetic biology community by improving interoperability.

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“…spectrometry combined with flow cytometry was used to assess P t (39). The NMR and protein concentration data were combined to construct binding isotherms (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Three 1-mL aliquots were taken from the in-cell culture for liquid chromatography mass spectrometry to determine protein concentration (39). The cells were pelleted at 2,000 × g for 20 min and washed three times with an in-cell NMR buffer (200 mM Hepes and 100 mM bis-Tris propane dissolved in 10% D 2 O, pH 7.6).…”

Section: Methodsmentioning

confidence: 99%

The intracellular environment affects protein–protein interactions

Speer

Zheng

Jiang

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Protein–protein interactions are essential for life but rarely thermodynamically quantified in living cells. In vitro efforts show that protein complex stability is modulated by high concentrations of cosolutes, including synthetic polymers, proteins, and cell lysates via a combination of hard-core repulsions and chemical interactions. We quantified the stability of a model protein complex, the A34F GB1 homodimer, in buffer, Escherichia coli cells and Xenopus laevis oocytes. The complex is more stable in cells than in buffer and more stable in oocytes than E. coli. Studies of several variants show that increasing the negative charge on the homodimer surface increases stability in cells. These data, taken together with the fact that oocytes are less crowded than E. coli cells, lead to the conclusion that chemical interactions are more important than hard-core repulsions under physiological conditions, a conclusion also gleaned from studies of protein stability in cells. Our studies have implications for understanding how promiscuous—and specific—interactions coherently evolve for a protein to properly function in the crowded cellular environment.

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Controlling and quantifying protein concentration in Escherichia coli

Cited by 11 publications

References 30 publications

High intracellular stability of the spidroin N‐terminal domain in spite of abundant amyloidogenic segments revealed by in‐cell hydrogen/deuterium exchange mass spectrometry

High intracellular stability of the spidroin N‐terminal domain in spite of abundant amyloidogenic segments revealed by in‐cell hydrogen/deuterium exchange mass spectrometry

FlopR: An Open Source Software Package for Calibration and Normalization of Plate Reader and Flow Cytometry Data

The intracellular environment affects protein–protein interactions

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