scholarly journals Multi-enzyme digestion FASP and the ‘Total Protein Approach’-based absolute quantification of the Escherichia coli proteome

2014 ◽  
Vol 109 ◽  
pp. 322-331 ◽  
Author(s):  
Jacek R. Wiśniewski ◽  
Dariusz Rakus
Keyword(s):  
Escherichia Coli ◽  
Total Protein ◽  
Absolute Quantification ◽  
Enzyme Digestion

scholarly journals LOCALIZATION OF MACROMOLECULES IN ESCHERICHIA COLI

1961 ◽  
Vol 9 (3) ◽  
pp. 555-565 ◽  
Author(s):  
Lucien G. Caro ◽  
Frederick Forro
Keyword(s):  
Escherichia Coli ◽  
Cross Sections ◽  
Short Pulse ◽  
Large Fraction ◽  
Enzyme Digestion ◽  
Morphological Data ◽  
Nuclear Region ◽  
Serial Sections ◽  
Short Pulses ◽  
E Coli

The distribution of RNA in cells of E. coli 15 T-U- labeled with uridine-H3 was studied by methods involving the analysis of radioautographic grain counts over random thin cross-sections and serial sections of the cells. The results were correlated with electron microscope morphological data. Fractionation and enzyme digestion studies showed that a large proportion of the label was found in RNA uracil and cytosine, the rest being incorporated as DNA cytosine. In fully labeled cells the distribution of label was found to be uniform throughout the cell. The situation remained unchanged when labeled cells were subsequently treated with chloramphenicol. When short pulses of label were employed a localization of a large proportion of the radioactivity became apparent. The nuclear region was identified as the site of concentration. Similar results were obtained when cells were exposed to much longer pulses of uridine-H3 in the presence of chloramphenicol. If cells were subjected to a short pulse of cytidine-H3, then allowed to grow for a while in unlabeled medium, the label, originally concentrated to some extent in the nuclear region, was found dispersed throughout the cell. The simplest hypothesis which accounts for these results is that a large fraction of the cell RNA is synthesized in a region in or near the nucleus and subsequently transferred to the cytoplasm.

Absolute quantification of the Kdp subunits of Escherichia coli by multiple reaction monitoring

PROTEOMICS ◽  
2014 ◽  
Vol 14 (13-14) ◽  
pp. 1630-1638 ◽  
Author(s):  
Kristin Surmann ◽  
Vera Laermann ◽  
Petra Zimmann ◽  
Karlheinz Altendorf ◽  
Elke Hammer
Keyword(s):  
Escherichia Coli ◽  
Multiple Reaction Monitoring ◽  
Absolute Quantification ◽  
Reaction Monitoring

Liquid chromatography coupled to multiple reaction monitoring (LC-MRM) for quantification of PD-L1 and PD1-signaling proteins in non-small cell lung carcinoma (NSCLC).

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e21040-e21040
Author(s):  
Vincent Lacasse ◽  
Rene Zahedi ◽  
Vincent Richard ◽  
Hangjun Wang ◽  
Georgia Mitsa ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Total Protein ◽  
Multiple Reaction Monitoring ◽  
Unmet Need ◽  
Predictive Biomarkers ◽  
Absolute Quantification ◽  
Protein Recovery ◽  
P Value ◽  
Fixation Time ◽  
Cell Lung Carcinoma

e21040 Background: Improving the predictive biomarkers arena of checkpoint inhibitors (CPIs) beyond PD-L1 immunohistochemistry (IHC) is one of the most important unmet need in NSCLC. Up to 50% of patients that show positive PD-L1 expression by IHC do not respond to anti-PD-L1 treatments, and patients with low/undetectable PD-L1 have significantly improved survival with CPI. Moreover, PD-L1 IHC is heterogeneous, can be affected by tissue fixation time and post-translational modifications like glycosylation. Also, multiple studies indicated that PD-L1 expression alone does not reliably reflect the immune status of the tumor, thus requiring the measurement of other members of the PD1 signalling pathway. Methods: To address these issues, we developed a multiplexed targeted mass spectrometry-based (MS) assay for the quantitation of protein members of the PD-1/PD-L1 axis in formalin fixed paraffin-embedded (FFPE) tissue.The effect of fixation time on protein recovery was determined using differentially fixed H1915 cells. Liquid chromatography (LC) coupled to MRM was used to develop a targeted assay for PD-L1, PD-1, PD-L2, NT5E, LCK and ZAP70. Results: After 30 minutes fixation 33.6 µg of protein were extracted per mg of FFPE H1915 cells, while protein recovery after 7 days was 55.8 µg/mg, with greater variability (18% and 28% CV, p-value = ns). The optimized LC-MRM method allows the quantitation of PD-L1 and PD-1 down to 23 amol on-column. We evaluated the utility of our MRM assays using the H1915 FFPE cells (PD-L1 3+ by IHC) and determined an endogenous concentration of PD-L1 and NT5E as being 33.2±0.1 amol/µg of total protein and 4.8±0.5 fmol/µg respectively. Noteworthy, a known glycosylation site of PD-L1 was quantified at 10.9±0.3 amol/µg of total protein (30% of total). As increases sensitivity was required, anti-peptide antibodies were generated against the 15 best peptides. We then evaluated this LC-MRM method using MDA-MB-436 cells with lower levels of PD-L1 protein assessed by IHC. Determination of endogenous PD-L1 concentration (3.0 amol/µg of total protein) was achieved using anti-peptide immuno-enrichment followed by MRM. Conclusions: We developed a fixation time independent extraction technique for FFPE and optimized a highly sensitive LC-MRM method that allows the absolute quantification of our targets. This proteomic workflow allows absolute quantification of the PD-1/PD-L1 axis from FFPE tissue using immuno-enrichment and a multiplexed LC-MRM method.

Characterization of 4 T1-like lytic bacteriophages that lyse Shiga-toxin Escherichia coli O157:H7

2012 ◽  
Vol 58 (7) ◽  
pp. 923-927 ◽  
Author(s):  
Yan D. Niu ◽  
Kim Stanford ◽  
Hans-W. Ackermann ◽  
Tim A. McAllister
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Enzyme Digestion ◽  
Average Multiplicity ◽  
Restriction Enzyme Digestion ◽  
Escherichia Coli O157 ◽  
Fecal Shedding ◽  
E Coli ◽  
Phage Types

Bacteriophages are associated with reduced fecal shedding of Shiga-toxin-producing Escherichia coli O157:H7 (STEC O157:H7) in cattle. Four phages exhibiting activity against 12 of 14 STEC O157:H7 strains, representing 11 common phage types, were isolated. Phages did not lyse non-O157 E. coli, with 11 of the 12 STEC strains exhibiting extreme susceptibility (average multiplicity of infection (MOI) = 0.0003−0.0007). All phages had icosahedral heads with tapered, noncontractile tails, a morphology indicative of T1-like Siphoviridae. Genome size of all phages was ∼44 kb, but EcoRІ or HindIII digestion profiles differed among phages. Based on restriction enzyme digestion profiles, phages AHP24, AHS24, and AHP42 were more related (66.7%−82.4%) to each other than to AKS96, while AHP24 and AHS24, isolated from the same feedlot pen, exhibited the highest identity (88.9%−92.3%). Phages AHP24 and AHS24 exhibited the broadest host range and strongest lytic activity against STEC O157:H7, making them strong candidates for biocontrol of this bacterium in cattle.

scholarly journals High-Level Production of Human Leptin by Fed-Batch Cultivation of Recombinant Escherichia coli and Its Purification

1999 ◽  
Vol 65 (7) ◽  
pp. 3027-3032 ◽  
Author(s):  
Ki Jun Jeong ◽  
Sang Yup Lee
Keyword(s):  
Escherichia Coli ◽  
Total Protein ◽  
Anion Exchange Chromatography ◽  
Batch Cultivation ◽  
Exchange Chromatography ◽  
Total Protein Content ◽  
Fed Batch ◽  
T7 Promoter ◽  
High Level ◽  
Level Production

ABSTRACT Human leptin is a 16-kDa (146-amino-acid) protein that is secreted from adipocytes and influences body weight homeostasis. In order to obtain high-level production of leptin, the human obesegene coding for leptin was expressed in Escherichia coliBL21(DE3) under the strong inducible T7 promoter. The recombinant leptin was produced as inclusion bodies in E. coli, and the recombinant leptin content was as high as 54% of the total protein content. For production of recombinant human leptin in large amounts, pH-stat fed-batch cultures were grown. Expression of leptin was induced at three different cell optical densities at 600 nm (OD600), 30, 90, and 140. When cells were induced at an OD600 of 90, the amount of leptin produced was 9.7 g/liter (37% of the total protein). After simple purification steps consisting of inclusion body isolation, denaturation and refolding, and anion-exchange chromatography, 144.9 mg of leptin that was more than 90% pure was obtained from a 50-ml culture, and the recovery yield was 41.1%.

scholarly journals A targeted proteomics toolkit for high-throughput absolute quantification of Escherichia coli proteins

2014 ◽  
Vol 26 ◽  
pp. 48-56 ◽  
Author(s):  
Tanveer S. Batth ◽  
Pragya Singh ◽  
Vikram R. Ramakrishnan ◽  
Mirta M.L. Sousa ◽  
Leanne Jade G. Chan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Absolute Quantification ◽  
Targeted Proteomics

scholarly journals Antibacterial Activity of Defensin PaDef from Avocado Fruit (Persea americanavar.drymifolia) Expressed in Endothelial Cells againstEscherichia coliandStaphylococcus aureus

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Jaquelina Julia Guzmán-Rodríguez ◽  
Rodolfo López-Gómez ◽  
Luis M. Suárez-Rodríguez ◽  
Rafael Salgado-Garciglia ◽  
Luis C. Rodríguez-Zapata ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Total Protein ◽  
Persea Americana ◽  
Alternative Strategies ◽  
Antibiotic Resistant ◽  
E Coli ◽  
Bovine Endothelial Cell ◽  
Avocado Fruit ◽  
Plant Defensins

Antimicrobial therapy is a useful tool to control infectious diseases in general and rising antibiotic resistant microorganisms in particular. Alternative strategies are desirable, and antimicrobial peptides (AMP) represent attractive control agents. Mexican avocado (Persea americanavar.drymifolia) is used in traditional medicine; however, the AMP production has not been reported in this plant. We obtained a cDNA library from avocado fruit and clone PaDef was identified, which has a cDNA (249 bp) encoding a protein (78 aa) homologous with plant defensins (>80%). We expressed the defensinPaDefcDNA (pBME3) in the bovine endothelial cell line BVE-E6E7. Polyclonal and clonal populations were obtained and their activity was evaluated againstEscherichia coli,Staphylococcus aureus, andCandida albicans.E. coliviability was inhibited with 100 μg/mL of total protein from clones (>55%). Also,S. aureusviability was inhibited from 50 μg/mL total protein (27–38%) but was more evident at 100 μg/mL (52–65%). This inhibition was higher than the effect showed by polyclonal population (~23%). Finally, we did not detect activity againstC. albicans. These results are the first report that shows antimicrobial activity of a defensin produced by avocado and suggest that this AMP could be used in the control of pathogens.

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