Cloning, expression and two-step purification of recombinant His-tag enhanced green fluorescent protein over-expressed in Escherichia coli

ABSTRACT Escherichia coli O157:H7 is a leading cause of food-borne illness. This human pathogen produces Shiga toxins (Stx1 and Stx2) which inhibit protein synthesis by inactivating ribosome function. The present study describes a novel cell-based assay to detect Stx2 and inhibitors of toxin activity. A Vero cell line harboring a destabilized variant (half-life, 2 h) of the enhanced green fluorescent protein (d2EGFP) was used to monitor the toxin-induced inhibition of protein synthesis. This Vero-d2EGFP cell line produced a fluorescent signal which could be detected by microscopy or with a plate reader. However, a greatly attenuated fluorescent signal was detected in Vero-d2EGFP cells that had been incubated overnight with either purified Stx2 or a cell-free culture supernatant from Stx1- and Stx2-producing E. coli O157:H7. Dose-response curves demonstrated that the Stx2-induced inhibition of enhanced green fluorescent protein fluorescence mirrored the Stx2-induced inhibition of overall protein synthesis and identified a picogram-per-milliliter threshold for toxin detection. To establish our Vero-d2EGFP assay as a useful tool for the identification of toxin inhibitors, we screened a panel of plant compounds for antitoxin activities. Fluorescent signals were maintained when Vero-d2EGFP cells were exposed to Stx1- and Stx2-containing medium in the presence of either grape seed or grape pomace extract. The antitoxin properties of the grape extracts were confirmed with an independent toxicity assay that monitored the overall level of protein synthesis in cells treated with purified Stx2. These results indicate that the Vero-d2EGFP fluorescence assay is an accurate and sensitive method to detect Stx2 activity and can be utilized to identify toxin inhibitors.

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The Mechanism of Chlorine Damage Using Enhanced Green Fluorescent Protein-Expressing Escherichia coli

Water ◽

10.3390/w11102156 ◽

2019 ◽

Vol 11 (10) ◽

pp. 2156

Author(s):

Mizozoe ◽

Otaki ◽

Aikawa

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Green Fluorescent Protein ◽

Cell Membrane ◽

Fluorescence Intensity ◽

Enhanced Green Fluorescent Protein ◽

Hypochlorous Acid ◽

Fluorescent Protein ◽

E Coli ◽

Green Fluorescent

This study investigated how chlorine inactivates and damages Escherichia coli cells. E. coli that had transformed to express enhanced green fluorescent protein (EGFP) at the cytoplasm was treated with chlorine. Damage to the cell membrane and cell wall was analyzed by measuring the fluorescence intensity of the leaked EGFP, then accounting for the fluorescence deterioration. At pH 7, E. coli was lethally damaged after treatment with chlorine, but significant leakage of EGFP was not observed. In contrast, significant leakage of EGFP was observed at pH 9, even though E. coli was not as inactivated as it was at pH 7. Flow cytometry was used to confirm the fluorescence intensity of the remaining EGFP inside the cells. No significant fluorescence loss was observed in the cells at pH 7. However, at pH 9, the fluorescence intensity in the cells decreased, indicating leakage of EGFP. These results suggest that hypochlorous acid inactivates E. coli without damaging its cell membrane and cell wall, whereas the hypochlorite ion inactivates E. coli by damaging its cell membrane and cell wall. It was possible to confirm the chlorine damage mechanism on E. coli by measuring the fluorescence intensity of the leaked EGFP.

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