In vitro stability and expression of green fluorescent protein under high pressure conditions

Ehrmann, Matthias A.; Scheyhing, Carsten H.; Vogel, Rudi F.

doi:10.1046/j.1472-765x.2001.00892.x

Cited by 44 publications

(49 citation statements)

References 16 publications

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“…The protein is in the shape of a cylinder named β-can, consisting of 11 strands of β-sheet with an α-helix inside, which protect the fluorophore [49,50]. GFP is quite thermostable [51], very resistant to chemical and pressure-induced denaturation [52][53][54].…”

Section: Fluospheres ® As a Low Pressure Calibrantmentioning

confidence: 99%

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Development of a low-pressure diamond anvil cell and analytical tools to monitor microbial activities in situ under controlled P and T

Oger

Daniel

Picard

2006

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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To cite this version:Philippe Oger, Isabelle Daniel, Aude Picard. Development of a low-pressure diamond anvil cell and analytical tools to monitor microbial activities in situ under controlled P and T. poger@ens-lyon.fr AbstractWe have designed a new low-pressure Diamond Anvil Cell (DAC), calibrated two novel pressure calibrants and validated the use of semi-quantitative Raman and Xray spectroscopies to monitor the fate of microbes, their metabolism or their cellular components under controlled pressures and temperatures in the 0.1-1.4 GPa and 20°-300°C P,T range. The lowpressure DAC has a 250-600 µm thick observation diamond window to allow for lower detection limits and improved microscopic imaging. This new design allows the determination of cellular growth parameters from automated image analysis, which can be correlated with the spectroscopic data obtained on metabolism, ensuring high quality data collection on microbial activity under pressure. The novel pressure sensors offer the ease of use of the well-known ruby scale, while being more sensitive and reacting to pressure variations instantaneously.

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Section: Fluospheres ® As a Low Pressure Calibrantmentioning

confidence: 99%

“…4) Pressure variations are immediately reported in the fluorescence spectrum. 5) The protein stability domain, which ranges up to at least 600 MPa [53,54], exceeds the pressure domain that is used in experiences performed with most live microorganisms or their cellular enzymatic machinery.…”

Section: Gfp As An Intracellular Pressure Calibrantmentioning

confidence: 99%

Development of a low-pressure diamond anvil cell and analytical tools to monitor microbial activities in situ under controlled P and T

Oger

Daniel

Picard

2006

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…The wide-range use of GFP is a consequence of its unique properties: reduced sensitivity to higher temperatures, detergents, alkaline pH, photobleaching, organic salts, chaotropic salts and many proteases [10]. EGFP (enhanced green fluorescent protein, a mutant version of GFP) has emerged as a powerful fluorescent label for quantitative fluorescence microscopy applications [11].…”

Section: Introductionmentioning

confidence: 99%

Heterologous Expression and Purification of Recombinant Proapoptotic Human Protein Smac/Diablo with EGFP as Fusion Partner

Salamon¹,

Miklóssy²,

Albert³

et al. 2017

Studia UBB Chemia

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ABSTRACT. New proteins as molecular targets in development of therapies are discovered every day. However, study of their interactions with other proteins or binding partners in complex cellular environments has its limits. Therefore, high-yield production of these proteins in heterologous systems is a valid necessity, while obtaining these proteins linked to suitable fluorescent markers represents a step ahead in protein-protein interaction studies and cellular or subcellular localization.In this study, we present production of human SMAC/Diablo recombinant protein with EGFP as a fusion partner. High-yield expression of the fusion protein was carried out in E. coli Rosetta™(DE3)pLysS strain, and an acceptable purity of the protein was obtained after affinity chromatography purification and gel filtration. The obtained protein can be further used in protein-protein interaction studies, whereas our method represents a cost-effective and efficient production method for EGFP-fused proteins, applicable for a number of therapeutically important polypeptides.

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“…Environmental stress encountered by E. coli in the human intestine or in food processing also induces Stx-prophages (18)(19)(20). Human neutrophils in the intestine release antibacterial molecules, including H 2 O 2 , which stimulate Stx and phage production (19).…”

mentioning

confidence: 99%

Induction of Shiga Toxin-Encoding Prophage by Abiotic Environmental Stress in Food

Fang

Mercer

McMullen

et al. 2017

Appl Environ Microbiol

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The prophage-encoded Shiga toxin is a major virulence factor in Stxproducing Escherichia coli (STEC). Toxin production and phage production are linked and occur after induction of the RecA-dependent SOS response. However, foodrelated stress and Stx-prophage induction have not been studied at the single-cell level. This study investigated the effects of abiotic environmental stress on stx expression by single-cell quantification of gene expression in STEC O104:H4 Δstx2::gfp:: amp r . In addition, the effect of stress on production of phage particles was determined. The lethality of stressors, including heat, HCl, lactic acid, hydrogen peroxide, and high hydrostatic pressure, was selected to reduce cell counts by 1 to 2 log CFU/ml. The integrity of the bacterial membrane after exposure to stress was measured by propidium iodide (PI). The fluorescent signals of green fluorescent protein (GFP) and PI were quantified by flow cytometry. The mechanism of prophage induction by stress was evaluated by relative gene expression of recA and cell morphology. Acid (pH Ͻ 3.5) and H 2 O 2 (2.5 mM) induced the expression of stx 2 in about 18% and 3% of the population, respectively. The mechanism of prophage induction by acid differs from that of induction by H 2 O 2 . H 2 O 2 induction but not acid induction corresponded to production of infectious phage particles, upregulation of recA, and cell filamentation. Pressure (200 MPa) or heat did not induce the Stx2-encoding prophage (Stx2-prophage). Overall, the quantification method developed in this study allowed investigation of prophage induction and physiological properties at the single-cell level. H 2 O 2 and acids mediate different pathways to induce Stx2-prophage.IMPORTANCE Induction of the Stx-prophage in STEC results in production of phage particles and Stx and thus relates to virulence as well as the transduction of virulence genes. This study developed a method for a detection of the induction of Stxprophages at the single-cell level; membrane permeability and an indication of SOS response to environmental stress were additionally assessed. H 2 O 2 and mitomycin C induced expression of the prophage and activated a SOS response. In contrast, HCl and lactic acid induced the Stx-prophage but not the SOS response. The lifestyle of STEC exposes the organism to intestinal and extraintestinal environments that impose oxidative and acid stress. A more thorough understanding of the influence of food processing-related stressors on Stx-prophage expression thus facilitates control of STEC in food systems by minimizing prophage induction during food production and storage.KEYWORDS single-cell detection, E. coli O104:H4, prophage induction, environmental stress, RecA-independent SOS response, membrane permeability, STEC, Shiga toxin, Shiga toxin-producing E. coli S higa toxin-producing Escherichia coli (STEC) causes the life-threatening hemolyticuremic syndrome (HUS), which is associated with acute renal failure and significant mortality (1, 2). Shiga toxins (Stx) include St...

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In vitro stability and expression of green fluorescent protein under high pressure conditions

Cited by 44 publications

References 16 publications

Development of a low-pressure diamond anvil cell and analytical tools to monitor microbial activities in situ under controlled P and T

Development of a low-pressure diamond anvil cell and analytical tools to monitor microbial activities in situ under controlled P and T

Heterologous Expression and Purification of Recombinant Proapoptotic Human Protein Smac/Diablo with EGFP as Fusion Partner

Induction of Shiga Toxin-Encoding Prophage by Abiotic Environmental Stress in Food

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