A versatile and economical method for the release of recombinant proteins from Escherichia coli by 1-propanol cell disruption

RSC Advances ◽  
2016 ◽  
Vol 6 (67) ◽  
pp. 62291-62297 ◽  
Author(s):  
Sewn Cen Lo ◽  
Ramakrishnan Nagasundara Ramanan ◽  
Beng Ti Tey ◽  
Weng Siang Tan ◽  
Pau Loke Show ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Recombinant Proteins ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Disruption ◽  
Economical Method ◽  
P Release ◽  
Green Fluorescent

Release of enhanced green fluorescent protein from Escherichia coli by 1-propanol cell disruption.

scholarly journals The Functional Quality of Soluble Recombinant Polypeptides Produced in Escherichia coli Is Defined by a Wide Conformational Spectrum

2008 ◽  
Vol 74 (23) ◽  
pp. 7431-7433 ◽  
Author(s):  
Mónica Martínez-Alonso ◽  
Nuria González-Montalbán ◽  
Elena García-Fruitós ◽  
Antonio Villaverde
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Recombinant Proteins ◽  
Fluorescent Protein ◽  
Native Structure ◽  
Functional Quality ◽  
Soluble Aggregates ◽  
Recombinant Polypeptides ◽  
Green Fluorescent

ABSTRACT We have observed that a soluble recombinant green fluorescent protein produced in Escherichia coli occurs in a wide conformational spectrum. This results in differently fluorescent protein fractions in which morphologically diverse soluble aggregates abound. Therefore, the functional quality of soluble versions of aggregation-prone recombinant proteins is defined statistically rather than by the prevalence of a canonical native structure.

scholarly journals Novel Cell-Based Method To Detect Shiga Toxin 2 from Escherichia coli O157:H7 and Inhibitors of Toxin Activity

2009 ◽  
Vol 75 (5) ◽  
pp. 1410-1416 ◽  
Author(s):  
Beatriz Quiñones ◽  
Shane Massey ◽  
Mendel Friedman ◽  
Michelle S. Swimley ◽  
Ken Teter
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Green Fluorescent Protein ◽  
Cell Line ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Grape Pomace ◽  
Response Curves ◽  
Fluorescent Signal ◽  
Green Fluorescent

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.

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

Water ◽  
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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