scholarly journals Characterization of a mammalian homolog of the Escherichia coli MutY mismatch repair protein.

1995 ◽  
Vol 15 (2) ◽  
pp. 989-996 ◽  
Author(s):  
J P McGoldrick ◽  
Y C Yeh ◽  
M Solomon ◽  
J M Essigmann ◽  
A L Lu
Keyword(s):  
Escherichia Coli ◽  
Specific Binding ◽  
Polyclonal Antibodies ◽  
Gradient Centrifugation ◽  
Immunoblot Analysis ◽  
Partial Purification ◽  
Phosphodiester Bond ◽  
E Coli ◽  
Nuclear Extracts ◽  
Calf Thymus

A protein homologous to the Escherichia coli MutY protein, referred to as MYH, has been identified in nuclear extracts of calf thymus and human HeLa cells. Western blot (immunoblot) analysis using polyclonal antibodies to the E. coli MutY protein detected a protein of 65 kDa in both extracts. Partial purification of MYH from calf thymus cells revealed a 65-kDa protein as well as a functional but apparently degraded form of 36 kDa, as determined by glycerol gradient centrifugation and immunoblotting with anti-MutY antibodies. Calf MYH is a DNA glycosylase that specifically removes mispaired adenines from A/G, A/7,8-dihydro-8-oxodeoxyguanine (8-oxoG or GO), and A/C mismatches (mismatches indicated by slashes). A nicking activity that is either associated with or copurified with MYH was also detected. Nicking was observed at the first phosphodiester bond 3' to the apurinic or apyrimidinic (AP) site generated by the glycosylase activity. The nicking activity on A/C mismatches was 30-fold lower and the activity on A/GO mismatches was twofold lower than that on A/G mismatches. No nicking activity was detected on substrates containing other selected mismatches or homoduplexes. Nicking activity on DNA containing A/G mismatches was inhibited in the presence of anti-MutY antibodies or upon treatment with potassium ferricyanide, which oxidizes iron-sulfur clusters. Gel shift analysis showed specific binding complex formation with A/G and A/GO substrates, but not with A/A, C.GO, and C.G substrates. Binding is sevenfold greater on A/GO substrates than on A/G substrates. The eukaryotic MYH may be involved in the major repair of both replication errors and oxidative damage to DNA, the same functions as those of the E. coli MutY protein.

scholarly journals Lectin and E. coli Binding to Carbohydrate-Functionalized Oligo(ethylene glycol)-Based Microgels: Effect of Elastic Modulus, Crosslinker and Carbohydrate Density

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 263
Author(s):  
Fabian Schröer ◽  
Tanja J. Paul ◽  
Dimitri Wilms ◽  
Torben H. Saatkamp ◽  
Nicholas Jäck ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Elastic Modulus ◽  
Ethylene Glycol ◽  
Concanavalin A ◽  
Specific Binding ◽  
Network Density ◽  
Carbohydrate Binding ◽  
E Coli ◽  
Surface Recognition ◽  
Strong Anchoring

The synthesis of carbohydrate-functionalized biocompatible poly(oligo(ethylene glycol) methacrylate microgels and the analysis of the specific binding to concanavalin A (ConA) and Escherichia coli (E. coli) is shown. By using different crosslinkers, the microgels’ size, density and elastic modulus were varied. Given similar mannose (Man) functionalization degrees, the softer microgels show increased ConA uptake, possibly due to increased ConA diffusion in the less dense microgel network. Furthermore, although the microgels did not form clusters with E. coli in solution, surfaces coated with mannose-functionalized microgels are shown to bind the bacteria whereas galactose (Gal) and unfunctionalized microgels show no binding. While ConA binding depends on the overall microgels’ density and Man functionalization degree, E. coli binding to microgels’ surfaces appears to be largely unresponsive to changes of these parameters, indicating a rather promiscuous surface recognition and sufficiently strong anchoring to few surface-exposed Man units. Overall, these results indicate that carbohydrate-functionalized biocompatible oligo(ethylene glycol)-based microgels are able to immobilize carbohydrate binding pathogens specifically and that the binding of free lectins can be controlled by the network density.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

scholarly journals Rhizobium (Sinorhizobium)meliloti phn Genes: Characterization and Identification of Their Protein Products

1999 ◽  
Vol 181 (2) ◽  
pp. 389-395 ◽  
Author(s):  
George F. Parker ◽  
Timothy P. Higgins ◽  
Timothy Hawkes ◽  
Robert L. Robson
Keyword(s):  
Escherichia Coli ◽  
Sinorhizobium Meliloti ◽  
Insertional Mutagenesis ◽  
Biochemical Characterization ◽  
Polyclonal Antibodies ◽  
E Coli ◽  
New Information ◽  
Phosphorus Sources ◽  
Carbon Phosphorus Lyase

ABSTRACT In Escherichia coli, the phn operon encodes proteins responsible for the uptake and breakdown of phosphonates. The C-P (carbon-phosphorus) lyase enzyme encoded by this operon which catalyzes the cleavage of C-P bonds in phosphonates has been recalcitrant to biochemical characterization. To advance the understanding of this enzyme, we have cloned DNA fromRhizobium (Sinorhizobium) melilotithat contains homologues of the E. coli phnG, -H, -I, -J, and -Kgenes. We demonstrated by insertional mutagenesis that the operon from which this DNA is derived encodes the R. meliloti C-P lyase. Furthermore, the phenotype of this phn mutant shows that the C-P lyase has a broad substrate specificity and that the organism has another enzyme that degrades aminoethylphosphonate. A comparison of the R. meliloti and E. coli phngenes and their predicted products gave new information about C-P lyase. The putative R. meliloti PhnG, PhnH, and PhnK proteins were overexpressed and used to make polyclonal antibodies. Proteins of the correct molecular weight that react with these antibodies are expressed by R. meliloti grown with phosphonates as sole phosphorus sources. This is the first in vivo demonstration of the existence of these hitherto hypothetical Phn proteins.

DNA Colony Hybridization Method Using Synthetic Oligonucleotides to Detect Enterotoxigenic Escherichia coli: Collaborative Study

1986 ◽  
Vol 69 (3) ◽  
pp. 531-536
Author(s):  
Walter E Hill ◽  
Barry A Wentz ◽  
William L Payne ◽  
James A Jagow ◽  
Gerald Zon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Dna ◽  
Collaborative Study ◽  
Dna Probes ◽  
Enterotoxigenic Escherichia Coli ◽  
Hybridization Probe ◽  
Colony Hybridization ◽  
E Coli ◽  
Calf Thymus ◽  
Recombinant Dna Techniques

Abstract The genes that encode several of the enterotoxins produced by Escherichia coli have been cloned by recombinant DNA techniques. When the nucleotide sequence of these genes is determined, defined sequence oligonucleotides that include a part of these genes may be synthesized. A 22-base DNA hybridization probe was produced for each of 2 heatstable E. coli enterotoxin (ST) genes: STH, from strains originally isolated from humans; and STP, from strains first found in pigs. For this study, 32P end-labeled DNA probes, sonicated calf thymus DNA, and 3 known and 20 unknown (10 ST-positive and 10 ST-negative) strains were sent to each of 23 collaborators. Cultures were spotted onto an agar-based medium and grown into colonies, which were transferred by blotting to cellulose filters, lysed by alkali and steam, and used for DNA colony hybridization with the ST DNA probes. Strains containing an ST gene were recognized as dark spots on an autoradiogram. Of the 460 samples analyzed, 440 (95.7%) were correctly classified by the collaborators. The method has been adopted official first action.

scholarly journals Genetic and Functional Analyses of the Conserved C-Terminal Core Domain of Escherichia coli FtsZ

1999 ◽  
Vol 181 (24) ◽  
pp. 7531-7544 ◽  
Author(s):  
Xiaolan Ma ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Immunoblot Analysis ◽  
Conserved Residues ◽  
Core Domain ◽  
Mutant Proteins ◽  
E Coli ◽  
Stable Mutant ◽  
Functional Analyses

ABSTRACT In Escherichia coli, FtsZ is required for the recruitment of the essential cell division proteins FtsA and ZipA to the septal ring. Several C-terminal deletions of E. coliFtsZ, including one of only 12 amino acids that removes the highly conserved C-terminal core domain, failed to complement chromosomalftsZ mutants when expressed on a plasmid. To identify key individual residues within the core domain, six highly conserved residues were replaced with alanines. All but one of these mutants (D373A) failed to complement an ftsZ chromosomal mutant. Immunoblot analysis demonstrated that whereas I374A and F377A proteins were unstable in the cell, L372A, D373A, P375A, and L378A proteins were synthesized at normal levels, suggesting that they were specifically defective in some aspect of FtsZ function. In addition, all four of the stable mutant proteins were able to localize and form rings at potential division sites in chromosomal ftsZ mutants, implying a defect in a function other than localization and multimerization. Because another proposed function of FtsZ is the recruitment of FtsA and ZipA, we tested whether the C-terminal core domain was important for interactions with these proteins. Using two different in vivo assays, we found that the 12-amino-acid truncation of FtsZ was defective in binding to FtsA. Furthermore, two point mutants in this region (L372A and P375A) showed weakened binding to FtsA. In contrast, ZipA was capable of binding to all four stable point mutants in the FtsZ C-terminal core but not to the 12-amino-acid deletion.

scholarly journals Anti-Escherichia coli Functionalized Silver-Doped Carbon Nanofibers for Capture of E. coli in Microfluidic Systems

Polymers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1117 ◽  
Author(s):  
Soshana Smith ◽  
Michael Delaney ◽  
Margaret Frey
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Carbon Nanofibers ◽  
Specific Binding ◽  
Base Material ◽  
Fiber Surface ◽  
Electrospinning Process ◽  
Silver Particles ◽  
Microfluidic Systems ◽  
E Coli

Silver-doped carbon nanofibers (SDCNF) are used as the base material for the selective capture of Escherichia coli in microfluidic systems. Fibers were spun in a glovebox with dry atmosphere maintained by forced dry air pumped through the closed environment. This affected the evaporation rate of the solvent during the electrospinning process and the distribution of silver particles within the fiber. Antibodies are immobilized on the surface of the silver-doped polyacrylonitrile (PAN) based carbon nanofibers via a three-step process. The negatively charged silver particles present on the surface of the nanofibers provide suitable sites for positively charged biotinylated poly-(L)-lysine-graft-poly-ethylene-glycol (PLL-g-PEG biotin) conjugate attachment. Streptavidin and a biotinylated anti-E. coli antibody were then added to create anti-E. coli surface functionalized (AESF) nanofibers. Functionalized fibers were able to immobilize up to 130 times the amount of E. coli on the fiber surface compared to neat silver doped fibers. Confocal images show E. coli remains immobilized on fiber mat surface after extensive rinsing showing the bacteria is not simply a result of non-specific binding. To demonstrate selectivity and functionalization with both gram negative and gram-positive antibodies, anti-Staphylococcus aureus surface functionalized (ASSF) nanofibers were also prepared. Experiments with AESF performed with Staphylococcus aureus (S. aureus) and ASSF with E. coli show negligible binding to the fiber surface showing the selectivity of the functionalized membranes. This surface functionalization can be done with a variety of antibodies for tunable selective pathogen capture.

scholarly journals Sodium Dodecyl Sulfate Hypersensitivity of clpP and clpB Mutants of Escherichia coli

2002 ◽  
Vol 68 (8) ◽  
pp. 4117-4121 ◽  
Author(s):  
Soumitra Rajagopal ◽  
Narasimhan Sudarsan ◽  
Kenneth W. Nickerson
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulfate ◽  
Uronic Acid ◽  
Sodium Dodecyl ◽  
Immunoblot Analysis ◽  
Wild Type ◽  
E Coli ◽  
Dodecyl Sulfate ◽  
Shock Proteins ◽  
Western Immunoblot Analysis

ABSTRACT We studied the hypersensitivity of clpP and clpB mutants of Escherichia coli to sodium dodecyl sulfate (SDS). Both wild-type E. coli MC4100 and lon mutants grew in the presence of 10% SDS, whereas isogenic clpP and clpB single mutants could not grow above 0.5% SDS and clpA and clpX single mutants could not grow above 5.0% SDS. For wild-type E. coli, cellular ClpP levels as determined by Western immunoblot analysis increased ca. sixfold as the levels of added SDS increased from 0 to 2%. Capsular colanic acid, measured as uronic acid, increased ca. sixfold as the levels of added SDS increased from 2 to 10%. Based on these findings, 3 of the 19 previously identified SDS shock proteins (M. Adamowicz, P. M. Kelley, and K. W. Nickerson, J. Bacteriol. 173:229-233, 1991) are tentatively identified as ClpP, ClpX, and ClpB.

scholarly journals Contribution of the Twin Arginine Translocation System to the Virulence of Enterohemorrhagic Escherichia coli O157:H7

2003 ◽  
Vol 71 (9) ◽  
pp. 4908-4916 ◽  
Author(s):  
Nathalie Pradel ◽  
Changyun Ye ◽  
Valérie Livrelli ◽  
Jianguo Xu ◽  
Bernard Joly ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Immunoblot Analysis ◽  
Vero Cells ◽  
Soft Agar ◽  
Enterohemorrhagic Escherichia Coli ◽  
E Coli ◽  
Twin Arginine Translocation ◽  
Tat System ◽  
K 12

ABSTRACT Shiga toxin-producing Escherichia coli O157:H7 is a major food-borne infectious pathogen. In order to analyze the contribution of the twin arginine translocation (TAT) system to the virulence of E. coli O157:H7, we deleted the tatABC genes of the O157:H7 EDL933 reference strain. The mutant displayed attenuated toxicity on Vero cells and completely lost motility on soft agar plates. Further analyses revealed that the ΔtatABC mutation impaired the secretion of the Shiga toxin 1 (Stx1) and abolished the synthesis of H7 flagellin, which are two major known virulence factors of enterohemorrhagic E. coli O157:H7. Expression of the EDL933 stxAB 1 genes in E. coli K-12 conferred verotoxicity on this nonpathogenic strain. Remarkably, cytotoxicity assay and immunoblot analysis showed, for the first time, an accumulation of the holotoxin complex in the periplasm of the wild-type strain and that a much smaller amount of StxA1 and reduced verotoxicity were detected in the ΔtatC mutant cells. Together, these results establish that the TAT system of E. coli O157:H7 is an important virulence determinant of this enterohemorrhagic pathogen.

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.

Overexpression of the ftsZ gene from Corynebacterium glutamicum (Brevibacterium lactofermentum) in Escherichia coli

2005 ◽  
Vol 51 (1) ◽  
pp. 85-89 ◽  
Author(s):  
María Pilar Honrubia-Marcos ◽  
Angelina Ramos ◽  
José A Gil
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Polyclonal Antibodies ◽  
T7 Promoter ◽  
Brevibacterium Lactofermentum ◽  
E Coli ◽  
Ftsz Protein ◽  
Pure Protein ◽  
Ftsz Gene ◽  
Dna Deletions

Our goal in this work was to overexpress the essential cell division FtsZ protein from Corynebacterium glutamicum (Brevibacterium lactofermentum) (FtsZCG) in Escherichia coli to produce anti-FtsZCG polyclonal antibodies. Previous results from our laboratory showed that ftsZCG was not expressed in E. coli in a sufficient amount to purify FtsZCG. However, when ftsZCG (without upstream sequences) was transcriptionally fused to the T7 promoter, different truncated FtsZCG proteins (28–32 kDa) were overexpressed in E. coli, and in all cases, stop codons were created because of DNA deletions or rearrangements. Nevertheless, we were able to overexpress and purify an N-terminally hexa-His-tagged FtsZCG from uninduced E. coli cells carrying a pET-28a(+) derivative, yielding about 5 mg of 98% pure protein per 100-mL culture.Key words: Brevibacterium lactofermentum, Corynebacterium glutamicum, FtsZ overexpresssion, hexa-His-tagged FtsZ.

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