scholarly journals Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Madison A. Smith ◽  
Jesica Gonzalez ◽  
Anjum Hussain ◽  
Rachel N. Oldfield ◽  
Kathryn A. Johnston ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Enzymatic Activity ◽  
Lysyl Oxidase ◽  
The Other ◽  
Glutathione S Transferase ◽  
E Coli ◽  
N Terminus ◽  
Low Solubility ◽  
Total Enzyme ◽  
Matrix Enzyme

Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5 mg for the Nus-A tagged enzyme (0.75 mg/L of media), 7.84 mg for the Trx tagged enzyme (3.92 mg/L of media), and 9.33 mg for the GST tagged enzyme (4.67 mg/L of media). Enzymatic activity was calculated to be 0.11 U/mg for the Nus-A tagged enzyme and 0.032 U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4 mM−1 cm−1. No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture.

scholarly journals The N Terminus of MinD Contains Determinants Which Affect Its Dynamic Localization and Enzymatic Activity

2004 ◽  
Vol 186 (21) ◽  
pp. 7175-7185 ◽  
Author(s):  
Jason Szeto ◽  
Sudeep Acharya ◽  
Nelson F. Eng ◽  
Jo-Anne R. Dillon
Keyword(s):  
Enzymatic Activity ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Conserved Residues ◽  
Dynamic Localization ◽  
Mutant Proteins ◽  
E Coli ◽  
N Terminus ◽  
Green Fluorescent

ABSTRACT MinD is involved in regulating the proper placement of the cytokinetic machinery in some bacteria, including Neisseria gonorrhoeae and Escherichia coli. Stimulation of the ATPase activity of MinD by MinE has been proposed to induce dynamic, pole-to-pole oscillations of MinD in E. coli. Here, we investigated the effects of deleting or mutating conserved residues within the N terminus of N. gonorrhoeae MinD (MinDNg) on protein dynamism, localization, and interactions with MinDNg and with MinENg. Deletions or mutations were generated in the first five residues of MinDNg, and mutant proteins were evaluated by several functional assays. Truncation or mutation of N-terminal residues disrupted MinDNg interactions with itself and with MinE. Although the majority of green fluorescent protein (GFP)-MinDNg mutants could still oscillate from pole to pole in E. coli, the GFP-MinDNg oscillation cycles were significantly faster and were accompanied by increased cytoplasmic localization. Interestingly, in vitro ATPase assays indicated that MinDNg proteins lacking the first three residues or with an I5E substitution possessed higher MinENg-independent ATPase activities than the wild-type protein. These results indicate that determinants found within the extreme N terminus of MinDNg are implicated in regulating the enzymatic activity and dynamic localization of the protein.

Three-Dimensional Reconstruction of Two Forms of the Chaperonin GRO EL

1990 ◽  
Vol 48 (1) ◽  
pp. 258-259
Author(s):  
J.L. Carrascosa ◽  
G. Abella ◽  
S. Marco ◽  
M. Muyal ◽  
J.M. Carazo
Keyword(s):  
Three Dimensional ◽  
The Other ◽  
Two Dimensional ◽  
Series Method ◽  
Three Dimensional Reconstruction ◽  
Structural Components ◽  
E Coli ◽  
Dimensional Reconstruction ◽  
Morphogenetic Factors ◽  
Tilt Series

Chaperonins are a class of proteins characterized by their role as morphogenetic factors. They trantsiently interact with the structural components of certain biological aggregates (viruses, enzymes etc), promoting their correct folding, assembly and, eventually transport. The groEL factor from E. coli is a conspicuous member of the chaperonins, as it promotes the assembly and morphogenesis of bacterial oligomers and/viral structures.We have studied groEL-like factors from two different bacteria:E. coli and B.subtilis. These factors share common morphological features , showing two different views: one is 6-fold, while the other shows 7 morphological units. There is also a correlation between the presence of a dominant 6-fold view and the fact of both bacteria been grown at low temperature (32°C), while the 7-fold is the main view at higher temperatures (42°C). As the two-dimensional projections of groEL were difficult to interprete, we studied their three-dimensional reconstruction by the random conical tilt series method from negatively stained particles.

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.

Antibacterial Activity of Thujaoccidentalis,ThujaorientalisandChamaecyparisobtusa

2017 ◽  
Vol 8 (03) ◽  
Author(s):  
Kyoung- Sun Seo ◽  
Seong Woo Jin ◽  
Seongkyu Choi ◽  
Kyeong Won Yun
Keyword(s):  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Antibacterial Agent ◽  
Methanol Extract ◽  
The Other ◽  
Diffusion Method ◽  
E Coli ◽  
Agar Diffusion ◽  
Agar Diffusion Method ◽  
Crude Methanol Extract

The antibacterial activity of three Cupressaceae plants (Thujaoccidentalis,ThujaorientalisandChamaecyparisobtusa) was tested against three bacteria using the agar diffusion method. The ether and ethylacetate fraction of crude methanol extract from the three plants showed potent antibacterial activity against the tested microorganisms. The result showed that Staphylococcus aureus revealed the most sensitivity among the tested bacteria. Thujaoccidentalisether fraction and Thujaorientalis hexane fraction exhibited the highest antibacterial activity against Staphylococcus aureus. E. coli was shown the highest MIC values compared to the other two tested bacteria, which indicates the lowest antibacterial activity against the bacterium. This study promises an interesting future for designing a potentially active antibacterial agent from the three Cupressaceae plants.

Assessment of the exposure of swimmers to microbiological contaminants in fresh waters

1997 ◽  
Vol 35 (11-12) ◽  
pp. 157-163 ◽  
Author(s):  
G. J. Medema ◽  
I. A. van Asperen ◽  
A. H. Havelaar
Keyword(s):  
Geometric Mean ◽  
Microbiological Quality ◽  
The Other ◽  
Pollution Level ◽  
Intestinal Infection ◽  
E Coli ◽  
Faecal Pollution ◽  
Run Off ◽  
Thermotolerant Coliforms ◽  
Mean Concentration

As part of a prospective cohort study among triathletes to determine a relationship between the microbiological quality of fresh bathing water and the risk of acquiring an intestinal infection, the exposure of the triathletes to microbiological contaminants was assessed. Waters were collected at seven triathlons (swimming course 1–1.5km) held in the summer of 1993 and 1994 to have a range of water qualities. All were influenced by sewage effluents, most also by agricultural run-off. Samples were collected several weeks before the event to establish a sampling programme (1993) and during the actual exposure of the triathletes (1993 and 1994) and examined for thermotolerant coliforms alone (samples preceding the event) and for E. coli, faecal enterococci, Staphylococcus aureus, F-specific RNAphages, enteroviruses (1993 and 1994) and for thermophilic Campylobacter, Salmonella, Aeromonas, Plesiomonas shigelloides and Pseudomonas aeruginosa (1993). The samples taken in the weeks before the exposure showed significant differences in thermotolerant coliform concentration between locations, depths and times. Also during swimmer exposure, significant differences occurred in microorganism levels at the different sampling points over the swimming course. As the triathletes swam as a group, they were exposed to approximately the same water at the same time. The geometric mean concentration was used to characterise each site. In the epidemiological study, the risk of an intestinal infection correlated with the concentration of thermotolerant coliforms and E. coli but not with the other parameters. The geometric mean concentration of thermotolerant coliforms at the triathlons ranged from 11–330/100mL and 54–1,200/100mL E. coli. Ranking of the seven sites by faecal pollution level, based on the geometric mean concentration of a faecal indicator, resulted in a different ranking for each indicator. At the fresh water sites studied, only the ratio between the geometric mean density of E. coli and thermotolerant coliforms was constant. The ratio between the other parameters related to faecal pollution (faecal enterococci, F-specific RNA phages, enteroviruses) varied considerably. Water quality standards relating to faecal pollution can only be based on parameters that show a significant correlation with risk of intestinal illness.

scholarly journals Comparative Study of Ag Nanostructures: Molecular Simulations, Electrochemical Behavior, and Antibacterial Effect

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Álvaro de Jesús Ruíz-Baltazar ◽  
Simón Yobanny Reyes-López ◽  
D. Larrañaga ◽  
R. Pérez
Keyword(s):  
Cyclic Voltammetry ◽  
Ethylene Glycol ◽  
Comparative Study ◽  
Sodium Borohydride ◽  
Electrochemical Behavior ◽  
Antibacterial Effect ◽  
Chemical Reduction ◽  
Molecular Simulations ◽  
The Other ◽  
E Coli

Nanoparticles of Ag with different sizes and structures were obtained and studied. Two methods for reductions of Ag ions were employed, chemical reduction by sodium borohydride and ethylene glycol. Cuboctahedral and icosahedral structures were obtained. Molecular simulations were carried out in order to evaluate the reactivity of both structures. On the other hand, the electrochemical activity and antibacterial effect (E. coli) of the cuboctahedral and icosahedral structures were measured experimentally. The results obtained by molecular simulation, cyclic voltammetry, and antibacterial effect were compared and discussed in this work.

scholarly journals Escherichia coli O157:H7 SURVIVAL IN TRADITIONAL AND LOW LACTOSE YOGURT DURING FERMENTATION AND COOLING PERIODS

2017 ◽  
Vol 18 (0) ◽  
Author(s):  
Camila Sampaio Cutrim ◽  
Raphael Ferreira de Barros ◽  
Robson Maia Franco ◽  
Marco Antonio Sloboda Cortez
Keyword(s):  
Escherichia Coli ◽  
The Other ◽  
Lactose Hydrolysis ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Gas Formation ◽  
Whole Milk ◽  
Milk Contamination ◽  
Different Types ◽  
Fermentation Period

Abstract The purpose of this study was to evaluate the behavior of E. coli O157:H7 during lactose hydrolysis and fermentation of traditional and low lactose yogurt. It also aimed to verify E. coli O157:H7 survival after 12 h of storage at 4 ºC ±1 ºC. Two different types of yogurts were prepared, two with whole milk and two with pre-hydrolyzed whole milk; in both groups one yogurt was inoculated with E. coli O157:H7 and the other one was not inoculated. The survival of E. coli and pH of yogurt were determined during fermentation and after 12-h refrigeration. The results showed that E. coli O157:H7 was able to grow during the fermentation period (from 4.34 log CFU.mL-1 to 6.13 log CFU.mL-1 in traditional yogurt and 4.34 log CFU.mL-1 to 6.16 log CFU.mL-1 in low lactose yogurt). The samples with E. coli O157:H7 showed gas formation and syneresis. Thus, E. coli O157:H7 was able to survive and grow during fermentation of traditional and low lactose yogurts affecting the manufacture technology. Moreover, milk contamination by E. coli before LAB addition reduces the growth of L. bulgaricus and S. thermophilus especially when associated with reduction of lactose content.

Species interactions in mixed-community crystalline biofilms on urinary catheters

2007 ◽  
Vol 56 (11) ◽  
pp. 1549-1557 ◽  
Author(s):  
Sarah M. Macleod ◽  
David J. Stickler
Keyword(s):  
Urinary Tract ◽  
Species Interactions ◽  
The Other ◽  
Experimental Investigations ◽  
Crystal Formation ◽  
Urinary Catheters ◽  
E Coli ◽  
Pure Cultures ◽  
Providencia Stuartii

Previous experimental investigations of the crystalline biofilms that colonize and block urinary catheters have focussed on their formation by pure cultures of Proteus mirabilis. In the urine of patients undergoing long-term catheterization, P. mirabilis is commonly found in mixed communities with other urinary tract pathogens. Little is known about the effect that the other species have on the rate at which P. mirabilis encrusts catheters. In the present study, a set of data on the nature of the bacterial communities on 106 catheter biofilms has been analysed and it was found that while species such as Providencia stuartii and Klebsiella pneumoniae were commonly associated with P. mirabilis, when Escherichia coli, Morganella morganii or Enterobacter cloacae were present, P. mirabilis was rarely or never found. The hypothesis that the absence of P. mirabilis from some biofilm communities could be due to its active exclusion by other species has also been examined. Experiments in laboratory models showed that co-infection of P. mirabilis with M. morganii, K. pneumoniae or E. coli had no effect on the ability of P. mirabilis to encrust and block catheters. Co-infection with Ent. cloacae or Pseudomonas aeruginosa, however, significantly increased the time that catheters took to block (P <0.05). The growth of Ent. cloacae, M. morganii, K. pneumoniae or E. coli in the model for 72 h prior to superinfection with P. mirabilis significantly delayed catheter blockage. In the case of Ent. cloacae, for example, the mean time to blockage was extended from 28.7 h to 60.7 h (P ≤0.01). In all cases, however, P. mirabilis was able to generate alkaline urine, colonize the biofilms, induce crystal formation and block the catheters. The results suggest that although there is a degree of antagonism between P. mirabilis and some of the other urinary tract organisms, the effects are temporary and whatever the pre-existing urinary microbiota, infection with P. mirabilis is thus likely to lead to catheter encrustation and blockage.

scholarly journals Mapping of a spectrin-binding domain of human erythrocyte membrane protein 4.2

2002 ◽  
Vol 364 (3) ◽  
pp. 841-847 ◽  
Author(s):  
Debabrata MANDAL ◽  
Prasun K. MOITRA ◽  
Joyoti BASU
Keyword(s):  
Erythrocyte Membrane ◽  
Synthetic Peptide ◽  
Membrane Skeleton ◽  
Band 3 Protein ◽  
Fusion Peptides ◽  
Human Erythrocyte Membrane ◽  
Glutathione S Transferase ◽  
Protein 4.2 ◽  
N Terminus ◽  
Direct Binding

Protein 4.2 is a major component of the red blood cell membrane skeleton. Deficiency of protein 4.2 is linked with a variety of hereditary haemolytic anaemias. However, the interactions of protein 4.2 with other proteins of the erythrocyte membrane remain poorly understood. The major membrane-binding site for protein 4.2 resides on the cytoplasmic domain of band 3. Protein 4.2 interacts directly with spectrin in solution, suggesting that it stabilizes interactions between the membrane skeleton and the erythrocyte membrane. A 30kDa polypeptide, with its N-terminus corresponding to amino acid residue 269, derived by partial proteolysis of protein 4.2, was found to interact with biotinylated spectrin in gel renaturation assays. A series of overlapping glutathione S-transferase fusion peptides were constructed, and an α-helical domain encompassing residues 470–492 was found to be instrumental in mediating protein 4.2—spectrin interactions. Direct binding of a synthetic peptide, with the sequence corresponding to residues 470–492, to spectrin and the ability of the peptide to inhibit spectrin binding of protein 4.2 confirmed that these residues are crucial in mediating protein 4.2—spectrin interactions.

scholarly journals Different expression levels of two KgmB-His fusion proteins

2005 ◽  
Vol 70 (12) ◽  
pp. 1401-1407 ◽  
Author(s):  
Sandra Markovic ◽  
Sandra Vojnovic ◽  
Milija Jovanovic ◽  
Branka Vasiljevic
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Kanamycin Resistance ◽  
Expression Vectors ◽  
E Coli ◽  
C Terminus ◽  
N Terminus ◽  
Different Levels ◽  
Streptomyces Tenebrarius

The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.

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