Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

1987 ◽  
Vol 45 ◽  
pp. 924-925
Author(s):  
F. A. Durum ◽  
R. G. Goldman ◽  
T. J. Bolling ◽  
M. F. Miller
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Recombinant Dna ◽  
Test System ◽  
Cell Protein ◽  
Gram Negative Bacteria ◽  
Cytoplasmic Inclusions ◽  
Isolation And Purification ◽  
E Coli ◽  
Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

scholarly journals The soluble Y115E–Y117E variant of human glutaminyl cyclase is a valid target for X-ray and NMR screening of inhibitors against Alzheimer disease

2015 ◽  
Vol 71 (8) ◽  
pp. 986-992 ◽  
Author(s):  
Flavio DiPisa ◽  
Cecilia Pozzi ◽  
Manuela Benvenuti ◽  
Matteo Andreini ◽  
Guido Marconi ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Expression System ◽  
Site Directed Mutagenesis ◽  
Nmr Screening ◽  
Glutaminyl Cyclase ◽  
X Ray ◽  
E Coli ◽  
Pure Protein ◽  
Recent Developments

Recent developments in molecular pathology and genetics have allowed the identification of human glutaminyl cyclase (hQC) among the abnormal proteins involved in many neurodegenerative disorders. Difficulties in obtaining large quantities of pure protein may limit the use of crystallographic screening for drug development on this target. Site-directed mutagenesis experiments have led to the identification of some solvent-exposed residues that are absolutely critical to achieve increased solubility and to avoid precipitation of the enzyme in inclusion bodies when expressed inEscherichia coli. The designed variant Y115E–Y117E has been found to be able to provide large amounts of monodisperse, pure hQC from anE. coliexpression system. To validate the use of the artificial construct as a target for large-scale X-ray and NMR screening campaigns in the search for new inhibitors of hQC, the X-ray crystal structures of the hQC Y115E–Y117E variant and of its adduct with the inhibitor PBD-150 were determined.

scholarly journals Highly Effective Renaturation of a Streptokinase fromStreptococcus pyogenesDT7 as Inclusion Bodies Overexpressed inEscherichia coli

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Sy Le Thanh Nguyen ◽  
Dinh Thi Quyen ◽  
Hong Diep Vu
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Biochemical Characterization ◽  
Host Cells ◽  
Scale Production ◽  
Peptide Fragments ◽  
E Coli ◽  
Gene Coding ◽  
Large Scale Production ◽  
Highly Effective

The streptokinase (SK) is emerging as an important thrombolytic therapy agent in the treatment of patients suffering from cardiovascular diseases. We reported highly effective renaturation of a SK fromS. pyogenessDT7 overexpressed inE. coli, purification, and biochemical characterization. A gene coding for the SK was cloned fromS. pyogenessDT7. Because accumulation of active SK is toxic to the host cells, we have expressed it in the form of inclusion bodies. The mature protein was overexpressed inE. coliBL21 DE3/pESK under the control of the strong promotertacinduced by IPTG with a level of 60% of the total cell proteins. The activity of the rSK, renatured in phosphate buffer supplemented with Triton X-100 and glycerol, was covered with up to 41 folds of its initial activity. The purified of protein was identified with MALDI-TOF mass spectrometry through four peptide fragments, which showed 100% identification to the corresponding peptides of the putative SK from GenBank. Due to overexpression and highly effective renaturation of large amounts of inclusion bodies, the recombinantE. coliBL21 DE3/pESK system could be potentially applied for large-scale production of SK used in the therapy of acute myocardial infarction.

scholarly journals Colicin-mediated transport of DNA through the iron transporter FepA

2021 ◽  
Author(s):  
Ruth Cohen-Khait ◽  
Ameya Harmalkar ◽  
Phuong Pham ◽  
Melissa N Webby ◽  
Nicholas G Housden ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Active Transport ◽  
Large Scale ◽  
Receptor Interaction ◽  
Cellular Receptor ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Colicin B

Colicins are protein antibiotics used by bacteria to eliminate competing Escherichia coli. A key event in the selective colicin function is a highly specific initial recognition step of an outer membrane (OM) receptor, which consequently allows the active transport of the colicin across the otherwise impervious OM. Though the colicin-receptor interaction is exclusive, the translocation process is likely to be universal as many receptors and colicins have surprisingly simi-lar 3D folds. Here, using a combination of photo-activated crosslinking, mass spectrometry, and structural modeling, we reveal how colicin B (ColB) associates with its OM receptor FepA. We demonstrate that complex formation is co-incident with a large-scale conformational change in the colicin. In vivo crosslinking experiments and further simula-tions of the translocation process indicate that part of the colicin engages active transport by disguising itself to part of the cellular receptor. Applying live-cell fluorescence imaging we were able to follow ColB into E. coli and localize it within the periplasm. Finally, we demonstrate that single-stranded DNA coupled to ColB is transported into the bacte-rial periplasm, emphasizing that the import routes of colicins can be exploited to carry large cargo molecules into Gram-negative bacteria.

Human glucose-dependent insulinotropic polypeptide (GIP) is an antimicrobial adjuvant re-sensitising multidrug-resistant Gram-negative bacteria

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Da’san M. M. Jaradat ◽  
Nehaya Al-Karablieh ◽  
Basmah H. M. Zaarer ◽  
Wenyi Li ◽  
Khalil K.Y. Saleh ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Low Concentrations ◽  
Bacterial Mutants ◽  
Drug Efflux Pumps ◽  
Gastrointestinal Peptides

Abstract Increasing antibiotic resistance in Gram-negative bacteria has mandated the development of both novel antibiotics and alternative therapeutic strategies. Evidence of interplay between several gastrointestinal peptides and the gut microbiota led us to investigate potential and broad-spectrum roles for the incretin hormone, human glucose-dependent insulinotropic polypeptide (GIP) against the Enterobacteriaceae bacteria, Escherichia coli and Erwinia amylovora. GIP had a potent disruptive action on drug efflux pumps of the multidrug resistant bacteria E. coli TG1 and E. amylovora 1189 strains. The effect was comparable to bacterial mutants lacking the inner and outer membrane efflux pump factor proteins AcrB and TolC. While GIP was devoid of direct antimicrobial activity, it has a potent membrane depolarizing effect, and at low concentrations, it significantly potentiated the activity of eight antibiotics and bile salt by reducing MICs by 4-8-fold in E. coli TG1 and 4-20-fold in E. amylovora 1189. GIP can thus be regarded as an antimicrobial adjuvant with potential for augmenting the available antibiotic arsenal.

scholarly journals Blocking monocyte transmigration in in vitro system by a human antibody scFv anti-CD99. Efficient large scale purification from periplasmic inclusion bodies in E. coli expression system

2014 ◽  
Vol 408 ◽  
pp. 35-45 ◽  
Author(s):  
Diego Moricoli ◽  
William Anthony Muller ◽  
Damiano Cosimo Carbonella ◽  
Maria Cristina Balducci ◽  
Sabrina Dominici ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Expression System ◽  
Human Antibody ◽  
In Vitro System ◽  
E Coli

Disinfection of Fruits with Activated Water

2019 ◽  
Vol 10 ◽  
pp. 1864-1872
Author(s):  
Prof. Teodora P. Popova
Keyword(s):  
Antimicrobial Activity ◽  
Aqueous Solutions ◽  
Antimicrobial Properties ◽  
The Other ◽  
Gram Negative Bacteria ◽  
High Antimicrobial Activity ◽  
Gram Negative ◽  
E Coli ◽  
Number Of Microorganisms ◽  
Lower Effect

The effect of ionized aqueous solutions (anolytes and catholyte) in the processing of fruits (cherries, morellos, and strawberries) for decontamination has been tested. Freshly prepared analytes and catholyte without the addition of salts were used, as well as stored for 7 months anolytes, prepared with 0.5% NaCl and a combination of 0.5% NaCl and 0.5% Na2CO3. The anolyte prepared with a combination of 0.5% NaCl and 0.5% Na2CO3, as well as the anolyte obtained with 0.5% NaCl, exhibit high antimicrobial activity against the surface microflora of strawberries, cherries, and sour cherries. They inactivate E. coli for 15 minutes. The other species of the fam. Enterobacteriaceae were also affected to the maximum extent, as is the total number of microorganisms, especially in cherries and sour cherries. Even stored for 7 months, they largely retain their antimicrobial properties. Anolyte and catholyte, obtained without the addition of salts, showed a lower effect on the total number of microorganisms, but had a significant effect on Gram-negative bacteria, and especially with regard to the sanitary indicative E. coli.

DECONTAMINATION OF VETERINARY SURVEILLANCE OBJECTS BY NEW GENERATION MEDICINE

1970 ◽  
pp. 64-67
Author(s):  
М. S. Saypullaev ◽  
А. U. Koychuev ◽  
Т. B. Mirzoeva
Keyword(s):  
Staphylococcus Aureus ◽  
Inhalation Exposure ◽  
Gram Negative Bacteria ◽  
Hazard Class ◽  
E Coli ◽  
Highly Efficient ◽  
Mucous Membranes ◽  
Environmentally Safe ◽  
New Generation ◽  
And Staphylococcus Aureus

The successful conduct of disinfection measures largely depends on the availability of veterinary practice a highly efficient, environmentally safe disinfectants. In this regard, finding new highly efficient disinfectant remains relevant. Studies found that the "Polied" (OOO "Razvitie XXI Vek, Russia) can be attributed to the highly efficient and environmentally friendly means. Solutions "Polied" have a high disinfectant activity against smooth and rough surfaces in the laboratory against gram-positive, gram-negative bacteria, mycobacteria and spores of microorganisms. Studies have established that solutions should be "Polied" obezzarajivatmi E. coli (EA 1257) concentrations of 0.1% on smooth surfaces and Staphylococcus aureus concentration of 0.05% in 1 hour from the calculation of 0.25-0.3 litres/m2. Disinfection of rough test surfaces against Escherichia coli and Staphylococcus aureus occurred after treatment with 0,3% solution of 3-hour exposure, at a rate of 0.5 l/m2. It was also found that 1.0% solution "Polied" fully obezzarazhivatel test the surface of mycobacteria (PCs-5) and at double the 0.6% concentration for 24 hours. Disinfection of rough test surfaces contaminated with spores of B. cereus (PCs 96) was achieved with a 4.0% solution at twice the irrigation rate of 0.5 l/m2 at an exposure of 24 hours. Toxicity solutions of the drug "Polied" refer to "moderate" threat (hazard class 3) and low-hazard substances (4 hazard class) when applied to the skin, mucous membranes of the eyes, and inhalation exposure on the respiratory system.

Enzymatic Synthesis of 15N-L-aspartic Acid Using Recombinant Aspartase from Escherichia Coli K12

2008 ◽  
Vol 59 (11) ◽  
Author(s):  
Iulia Lupan ◽  
Sergiu Chira ◽  
Maria Chiriac ◽  
Nicolae Palibroda ◽  
Octavian Popescu
Keyword(s):  
Amino Acids ◽  
Aspartic Acid ◽  
Enzymatic Synthesis ◽  
Large Scale ◽  
Recombinant Dna ◽  
Industrial Enzymes ◽  
Recombinant Dna Technology ◽  
Bacterial Fermentation ◽  
Natural Protein ◽  
Novel Method

Amino acids are obtained by bacterial fermentation, extraction from natural protein or enzymatic synthesis from specific substrates. With the introduction of recombinant DNA technology, it has become possible to apply more rational approaches to enzymatic synthesis of amino acids. Aspartase (L-aspartate ammonia-lyase) catalyzes the reversible deamination of L-aspartic acid to yield fumaric acid and ammonia. It is one of the most important industrial enzymes used to produce L-aspartic acid on a large scale. Here we described a novel method for [15N] L-aspartic synthesis from fumarate and ammonia (15NH4Cl) using a recombinant aspartase.

Identification of N-Substituted Triazolo-azetidines as Novel Antibacterials using pDualrep2 HTS Platform

2019 ◽  
Vol 22 (5) ◽  
pp. 346-354
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Large Scale ◽  
Underlying Mechanism ◽  
Luciferase Assay ◽  
Reporter System ◽  
E Coli ◽  
Starting Point ◽  
High Concentrations ◽  
Mic Value

Aim and Objective: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. Materials and Methods: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. Results: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. Conclusion: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.

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