Cloning and expression of a functional rat liver D-β-hydroxybutyrate dehydrogenase-β-galactosidase fusion protein in Escherichia coli

1991 ◽  
Vol 69 (9) ◽  
pp. 670-673
Author(s):  
Sharon Churchill ◽  
Perry Churchill
Keyword(s):  
Escherichia Coli ◽  
Rat Liver ◽  
Polyclonal Antibodies ◽  
Cloning And Expression ◽  
Dependent Manner ◽  
Bacteriophage Λ ◽  
Expression Library ◽  
Expression Plasmid ◽  
E Coli ◽  
Dose Dependent

A rat liver bacteriophage λ expression library was probed using polyclonal antibodies raised to purified rat liver D-β-hydroxybutyrate dehydrogenase (BDH). A clone was selected that contained a 1.2-kb insert. The insert placed in an expression plasmid was utilized to transform Escherichia coli. These cells were shown to possess phosphatidylcholine-dependent BDH activity. Cells transformed with only the plasmid had no detectable BDH activity in the presence of phosphatidylcholine. The expressed activity in E. coli could be inhibited in a dose-dependent manner by BDH antiserum.Key words: D-β-hydroxybutyrate dehydrogenase, cloning, expression.

scholarly journals Intracellular Free Iron and Its Potential Role in Ultrahigh-Pressure-Induced Inactivation of Escherichia coli

2012 ◽  
Vol 79 (2) ◽  
pp. 722-724 ◽  
Author(s):  
Yuan Yan ◽  
Joy G. Waite-Cusic ◽  
Periannan Kuppusamy ◽  
Ahmed E. Yousef
Keyword(s):  
Escherichia Coli ◽  
Iron Chelator ◽  
Ultrahigh Pressure ◽  
Dependent Manner ◽  
Paramagnetic Resonance ◽  
Free Iron ◽  
Content Type ◽  
E Coli ◽  
Electron Paramagnetic ◽  
Dose Dependent

ABSTRACTIntracellular free iron ofEscherichia coliwas determined by whole-cell electron paramagnetic resonance spectrometry. Ultrahigh pressure (UHP) increased both intracellular free iron and cell lethality in a pressure-dose-dependent manner. The iron chelator 2,2′-dipyridyl protected cells against UHP treatments. A mutation that produced iron overload conditions sensitizedE. colito UHP treatment.

scholarly journals The Escherichia coli Common Pilus and the Bundle-Forming Pilus Act in Concert during the Formation of Localized Adherence by Enteropathogenic E. coli

2009 ◽  
Vol 191 (11) ◽  
pp. 3451-3461 ◽  
Author(s):  
Zeus Saldaña ◽  
Ayşen L. Erdem ◽  
Stephanie Schüller ◽  
Iruka N. Okeke ◽  
Mark Lucas ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Triple Mutant ◽  
E Coli ◽  
Accessory Factor ◽  
Isogenic Mutants ◽  
Dose Dependent ◽  
Background Expression

ABSTRACT Although the bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) mediates microcolony formation on epithelial cells, the adherence of BFP-deficient mutants is significantly abrogated, but the mutants are still adherent due to the presence of intimin and possibly other adhesins. In this study we investigated the contribution of the recently described E. coli common pilus (ECP) to the overall adherence properties of EPEC. We found that ECP and BFP structures can be simultaneously observed in the course (between zero time and 7 h during infection) of formation of localized adherence on cultured epithelial cells. These two pilus types colocalized at different levels of the microcolony topology, tethering the adhering bacteria. No evidence of BFP disappearance was found after prolonged infection. When expressed from a plasmid present in nonadherent E. coli HB101, ECP rendered this organism highly adherent at levels comparable to those of HB101 expressing the BFP. Purified ECP bound in a dose-dependent manner to epithelial cells, and the binding was blocked with anti-ECP antibodies, confirming that the pili possess adhesin properties. An ECP mutant showed only a modest reduction in adherence to cultured cells due to background expression levels of BFP and intimin. However, isogenic mutants not expressing EspA or BFP were significantly less adherent when the ecpA gene was also deleted. Furthermore, a ΔespA ΔecpA double mutant (unable to translocate Tir and to establish intimate adhesion) was at least 10-fold less adherent than the ΔespA and ΔecpA single mutants, even in the presence of BFP. A Δbfp ΔespA ΔecpA triple mutant showed the least adherence compared to the wild type and all the isogenic mutant strains tested, suggesting that ECP plays a synergistic role in adherence. Our data indicate that ECP is an accessory factor that, in association with BFP and other adhesins, contributes to the multifactorial complex interaction of EPEC with host epithelial cells.

scholarly journals Inhibition of Escherichia coli-Induced Meningitis by Carboxyfullerence

1999 ◽  
Vol 43 (9) ◽  
pp. 2273-2277 ◽  
Author(s):  
Nina Tsao ◽  
Puthuparampil P. Kanakamma ◽  
Tien-Yau Luh ◽  
Chen-Kung Chou ◽  
Huan-Yao Lei
Keyword(s):  
Escherichia Coli ◽  
Water Soluble ◽  
Dependent Manner ◽  
Neutrophilic Infiltration ◽  
Necrosis Factor Alpha ◽  
E Coli ◽  
Brain Barrier Permeability ◽  
Factor Alpha ◽  
Dose Dependent ◽  
Necrosis Factor

ABSTRACT The effect of a water-soluble malonic acid derivative of carboxyfullerence (C60) against Escherichia coli-induced meningitis was tested. C60 can protect the mice from E. coli-induced death in a dose-dependent manner. C60 administered intraperitoneally as late as 9 h after E. coliinjection was still protective. The C60-treated mice had less tumor necrosis factor alpha and interleukin-1β production by staining of brain tissue compared to the levels of production for nontreated mice. The E. coli-induced increases in blood-brain barrier permeability and inflammatory neutrophilic infiltration were also inhibited. These data suggest that C60 is a potentially therapeutic agent for bacterial meningitis.

scholarly journals Inhibitory Effect of Heterologous Ribosome Recycling Factor on Growth of Escherichia coli

2000 ◽  
Vol 182 (21) ◽  
pp. 6154-6160 ◽  
Author(s):  
Kenji Atarashi ◽  
Akira Kaji
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
Thermotoga Maritima ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Ribosome Recycling Factor ◽  
E Coli ◽  
Genes Encoding ◽  
Dose Dependent

ABSTRACT Ribosome recycling factor (RRF) of Thermotoga maritimawas expressed in Escherichia coli from the cloned T. maritima RRF gene and purified. Expression of T. maritima RRF inhibited growth of the E. coli host in a dose-dependent manner, an effect counteracted by the overexpression of E. coli RRF. T. maritima RRF also inhibited the E. coli RRF reaction in vitro. Genes encoding RRFs fromStreptococcus pneumoniae and Helicobacter pylori have been cloned, and they also impair growth of E. coli, although the inhibitory effect of these RRFs was less pronounced than that of T. maritima RRF. The amino acid sequence at positions 57 to 62, 74 to 78, 118 to 122, 154 to 160, and 172 to 176 in T. maritima RRF differed totally from that ofE. coli RRF. This suggests that these regions are important for the inhibitory effect of heterologous RRF. We further suggest that bending and stretching of the RRF molecule at the hinge between two domains may be critical for RRF activity and therefore responsible forT. maritima RRF inhibition of the E. coli RRF reaction.

scholarly journals Host Protein Binding and Adhesive Properties of H6 and H7 Flagella of Attaching and Effacing Escherichia coli

2007 ◽  
Vol 189 (20) ◽  
pp. 7426-7435 ◽  
Author(s):  
Ayşen L. Erdem ◽  
Fabiola Avelino ◽  
Juan Xicohtencatl-Cortes ◽  
Jorge A. Girón
Keyword(s):  
Escherichia Coli ◽  
Host Protein ◽  
Matrix Proteins ◽  
Dependent Manner ◽  
Host Proteins ◽  
Adhesive Properties ◽  
E Coli ◽  
Mucosal Surfaces ◽  
Dose Dependent ◽  
Type Strains

ABSTRACT It had been suggested that the flagella of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) might contribute to host colonization. In this study, we set out to investigate the adhesive properties of H7 and H6 flagella. We studied the abilities of EHEC EDL933 (O157:H7) and EPEC E2348/69 (O127:H6) flagella to bind to bovine mucus, host proteins such as mucins, and extracellular matrix proteins. Through several approaches, we found that H6 and H7 flagella and their flagellin monomers bind to mucins I and II and to freshly isolated bovine mucus. A genetic approach showed that EHEC and EPEC fliC deletion mutants were significantly less adherent to bovine intestinal tissue than the parental wild-type strains. In addition, we found that EPEC bacteria and H6 flagella, but not EHEC, bound largely, in a dose-dependent manner, to collagen and to a lesser extent to laminin and fibronectin. We also report that EHEC O157:H7 strains agglutinate rabbit red blood cells via their flagella, a heretofore unknown phenotype in this pathogroup. Collectively, our data demonstrate that the H6 and H7 flagella possess adhesive properties, particularly the ability to bind mucins, that may contribute to colonization of mucosal surfaces.

scholarly journals Inhibition of Complement and CD14 Attenuates the Escherichia coli-Induced Inflammatory Response in Porcine Whole Blood

2008 ◽  
Vol 77 (2) ◽  
pp. 725-732 ◽  
Author(s):  
Ebbe Billmann Thorgersen ◽  
Anne Pharo ◽  
Karin Haverson ◽  
Anne K. Axelsen ◽  
Peter Gaustad ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Inflammatory Response ◽  
Proinflammatory Cytokines ◽  
Whole Blood ◽  
Innate Immune ◽  
Dependent Manner ◽  
Bacterial Clearance ◽  
E Coli ◽  
Tnf Α ◽  
Dose Dependent

ABSTRACT The innate immune response is a double-edged sword in systemic inflammation and sepsis. Uncontrolled or inappropriate activation can damage and be lethal to the host. Several studies have investigated inhibition of downstream mediators, including tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). Emerging evidence indicates that upstream inhibition is a better therapeutic approach for attenuating damaging immune activation. Therefore, we investigated inhibition of two central innate immune pathways, those of complement and CD14/Toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD-2), in a porcine in vitro model of Escherichia coli-induced inflammation. Porcine whole blood anticoagulated with lepuridin, which did not interfere with the complement system, was incubated with E. coli lipopolysaccharide (LPS) or whole bacteria. Inhibitors of complement and CD14 and thus the LPS CD14/TLR4/MD-2 receptor complex were tested to investigate the effect on the inflammatory response. A broad range of inflammatory readouts were used to monitor the effect. Anti-CD14 was found to saturate the CD14 molecule on granulocytes and completely inhibited LPS-induced proinflammatory cytokines in a dose-dependent manner. Anti-CD14 significantly reduced the levels of the E. coli-induced proinflammatory cytokines TNF-α and IL-1β, but not IL-8, in a dose-dependent manner. No effect on bacterial clearance was seen. Vaccinia complement control protein and smallpox inhibitor of complement enzymes, two Orthopoxvirus-encoded complement inhibitors, completely inhibited complement activation. Furthermore, these agents almost completely inhibited the expression of wCD11R3, which is associated with CD18 as a β2 integrin, on porcine granulocytes and decreased IL-8 levels significantly in a dose-dependent manner. As expected, complement inhibition reduced bacterial clearance. We conclude that inhibition of complement and CD14 attenuates E. coli-induced inflammation and might be used as a therapeutic regimen in gram-negative sepsis along with appropriate treatment with antibiotics.

scholarly journals Effect of polyethylene glycol on growth of Escherichia coli DH5α and Bacillus subtilis NRS-762

2020 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Polyethylene Glycol ◽  
Optical Density ◽  
Culture Broth ◽  
Dependent Manner ◽  
Biomass Formation ◽  
E Coli ◽  
Toxicity Effect ◽  
Dose Dependent

AbstractPolyethylene glycol is commonly used in fermentation as an anti-foam for preventing the rise of foam to the top plate of the bioreactor, which increases contamination risk. However, its potential toxicity to growth of various microorganisms is not well understood at the species and strain level. Hence, the objective of this study was to understand the impact of different concentrations of polyethylene glycol at the 1, 5 and 10 g/L level on the aerobic growth of Escherichia coli DH5α and Bacillus subtilis NRS-762 in LB Lennox medium in shake flasks. Experiment results revealed that polyethylene glycol (PEG) (molecular weight ∼8000 Da), at all concentrations tested, did not affect biomass formation and metabolism in E. coli DH5α at 37 °C. This came about through the observation of similar maximal optical density obtained during growth of E. coli DH5α under differing concentrations of PEG. Furthermore, the anti-foam did not affect the pH profile. On the other hand, PEG did exhibit some toxicity towards the growth of B. subtilis NRS-762 in LB Lennox medium. Specifically, maximal optical density obtained decline with higher exposure to PEG in a concentration dependent manner, up to a threshold concentration of 5 g/L. For example, maximal optical density obtained in B. subtilis NRS-762 without addition of PEG was 4.4, but the value obtained with 1 g/L of the anti-foam decreased to 4.1 and a further 3.8 on exposure to 5 g/L and 10 g/L PEG. pH variation in culture broth, however, told a different story, where the profiles for exposure to PEG at all concentrations coincide with each other and was similar to the one without exposure to the anti-foam; thereby, suggesting that metabolic processes in B. subtilis NRS-762 were not significantly affected by exposure to PEG. Collectively, PEG anti-foam exerted species-specific toxicity effect on biomass formation, and possibly metabolism. The latter may not be sufficiently significant to affect the types of metabolites secreted by the bacterium, and thus, be detected by measurement of pH of culture broth. E. coli DH5α was better able to cope with PEG at all concentrations compared to B. subtilis NRS-762, which showed dose-dependent toxicity effect on biomass formation.HighlightsPolyethylene glycol (molecular weight ∼ 8000 Da) did not affect aerobic growth of Escherichia coli DH5α at 37 °C in LB medium at all concentrations tested: 0, 1, 5, 10 g/L.Growth curves of the bacterium at different concentrations of polyethylene glycol (PEG) coincided with each other.Similar pH profiles were also obtained for E. coli DH5α growth in LB medium with different PEG concentrations.However, PEG exerted toxicity effect on Bacillus subtilis NRS-762 during growth in LB medium at 30 °C, with reduction of biomass formation in a dose dependent manner.Similar to the case for E. coli DH5α. pH profiles of B. subtilis NRS-762 coincided with each other irrespective of the concentrations of PEG used.

scholarly journals On-column Refolding of an Insoluble His6-tagged Recombinant EC-SOD Overexpressed in Escherichia coli

2005 ◽  
Vol 37 (4) ◽  
pp. 265-269 ◽  
Author(s):  
Xi-Qiang Zhu ◽  
Su-Xia Li ◽  
Hua-Jun He ◽  
Qin-Sheng Yuan
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Mass Ratio ◽  
Chain Reaction ◽  
Expression Plasmid ◽  
Protein Subunits ◽  
E Coli ◽  
Metal Affinity ◽  
Polymerase Chain ◽  
Escherichia Coli Expression

Abstract The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a(+) and transformed into E. coli BL21(DE3). The corresponding protein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactive inclusion bodies. This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4:1. The purified enzyme was active, showing that at least part of the protein was properly refolded. The protein was made concentrated by ultrafiltration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A280 profile. Based on the results of electrophoresis, we concluded that the two fractions were formed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of the dimer was much higher than that of the single monomer. The special activities of the two fractions were found to be 3475 U/mg protein and 510 U/mg protein, respectively.

scholarly journals Escherichia coli as a genetic tool

1985 ◽  
Vol 95 (3) ◽  
pp. 611-618
Author(s):  
Naomi Datta
Keyword(s):  
Escherichia Coli ◽  
Academic Research ◽  
Genetically Engineered ◽  
Cloning And Expression ◽  
Biological Research ◽  
New Techniques ◽  
E Coli ◽  
Genetic Tool ◽  
The Past ◽  
Made In

SUMMARYThe study of Escherichia coli and its plasmids and bacteriophages has provided a vast body of genetical information, much of it relevant to the whole of biology. This was true even before the development of the new techniques, for cloning and analysing DNA, that have revolutionized biological research during the past decade. Thousands of millions of dollars are now invested in industrial uses of these techniques, which all depend on discoveries made in the course of academic research on E. coli. Much of the background of knowledge necessary for the cloning and expression of genetically engineered information, as well as the techniques themselves, came from work with this organism.

Cloning and expression of Xylella fastidiosa antigens in Escherichia coli and Erwinia stewartii

1989 ◽  
Vol 35 (4) ◽  
pp. 487-491 ◽  
Author(s):  
Paul H. Goodwin
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Genomic Dna ◽  
Xylella Fastidiosa ◽  
Gene Bank ◽  
Cloning And Expression ◽  
Western Blots ◽  
E Coli ◽  
Cosmid Vector ◽  
Erwinia Stewartii

Xylella fastidiosa DNA, partially digested with Sau3A, was ligated into the cosmid vector, pUCD615. Approximately 4500 ampicillin-resistant Escherichia coli colonies were obtained. The frequency of complementation of leucine auxotrophy in transfected E. coli indicated that the cosmid gene bank was representative of X. fastidiosa genomic DNA. Colonies were lysed directly onto nitrocellulose membranes using a thermo-inducible λ lysogen and screened for expression of X. fastidiosa antigens. Approximately 16.5% of a random sample of clones were found to express X. fastidiosa antigens as determined by Western blots. These proteins comigrated with proteins of X. fastidiosa and ranged in molecular weight from 10 000 to 160 000. Conjugation of several of the plasmids into Erwinia stewartii resulted in expression of the similar molecular weight cloned proteins with similar levels of expression as in E. coli.Key words: Xylella fastidiosa, Pierce's disease, immunological clone screening, thermo-inducible lysogeny.

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