Search results for "Erich"
showing 10 items of 805 documents
Sequence-specific and DNA structure-dependent interactions of Escherichia coli MutS and human p53 with DNA
2013
Many proteins involved in DNA repair systems interact with DNA that has structure altered from the typical B-form helix. Using magnetic beads to immobilize DNAs containing various types of structures, we evaluated the in vitro binding activities of two well-characterized DNA repair proteins, Escherichia coli MutS and human p53. E. coli MutS bound to double-stranded DNAs, with higher affinity for a G/T mismatch compared to a G/A mismatch and highest affinity for larger non-B-DNA structures. E. coli MutS bound best to DNA between pH 6 and 9. Experiments discriminated between modes of p53-DNA binding, and increasing ionic strength reduced p53 binding to nonspecific double-stranded DNA, but had…
High yield recombinant production of a self-assembling polycationic peptide for silica biomineralization.
2015
We report the recombinant bacterial expression and purification at high yields of a polycationic oligopeptide, P5S3. The sequence of P5S3 was inspired by a diatom silaffin, a silica precipitating peptide. Like its native model, P5S3 exhibits silica biomineralizing activity, but furthermore has unusual self-assembling properties. P5S3 is efficiently expressed in Escherichia coli as fusion with ketosteroid isomerase (KSI), which causes deposition in inclusion bodies. After breaking the fusion by cyanogen bromide reaction, P5S3 was purified by cation exchange chromatography, taking advantage of the exceptionally high content of basic amino acids. The numerous cationic charges do not prevent, b…
Enzymatic activity of circular sortase A under denaturing conditions: An advanced tool for protein ligation
2014
Abstract Staphylococcus aureus sortase A is a transpeptidase that is extensively used in various protein research applications. Sortase A is highly selective and does not require any cofactors for the catalysis of protein ligation and, importantly, can be produced in high yields. However, the primary disadvantage of this transpeptidase is its inability to access the recognition site within the highly structured regions of folded substrates. To overcome this problem, we developed an Escherichia coli expression system that produces milligram quantities of circularly closed sortase A; efficient enzyme cyclization was achieved by Synechocystis sp. PCC6803 intein-mediated post-translational spli…
Polymer-induced phase separation in Escherichia coli suspensions
2010
We studied aggregation and phase separation in suspensions of de-flagellated Escherichia coli (AB1157) in phosphate buffer induced by the anionic polyelectrolyte sodium polystyrene sulfonate. We also performed Monte Carlo simulations of this system based on the Asakura–Oosawa model of colloid–polymer mixtures. The results of these simulations, as well as comparison with previous work on synthetic colloid–polymer mixtures, demonstrate that the role of the polymer is to cause a depletion attraction between the E. coli cells. The implication of these results for understanding the role of (predominantly anionic) extracellular polymeric substances (EPS) secreted by bacteria in various natural ph…
Production of soluble eukaryotic recombinant proteins in E. coli is favoured in early log-phase cultures induced at low temperature
2013
Abstract Background Producing recombinant plant proteins expressed in Escherichia coli produce in high yields and in a soluble and functional form can be difficult. Under overexpression conditions, proteins frequently accumulate as insoluble aggregates (inclusion bodies) within the producing bacteria. We evaluated how the initial culture density, temperature and duration of the expression stage affect the production of some eukaryotic enzymes in E. coli. Findings A high yield of active soluble proteins was obtained by combining early-log phase cultures and low temperatures for protein induction. When IPTG was added at OD600 = 0.1 and cultures were maintained at 4°C for 48-72 h, the soluble …
Biosilica-based immobilization strategy for label-free OWLS sensors
2013
Abstract In the last years, a new group of enzymes, so-called silicateins, have been identified and characterized, which form the axial filaments of the spicules of the siliceous sponges, consisting of amorphous silica. Silicateins are able to catalyze the polycondensation and deposition of silica at mild conditions (low temperature and physiological pH). By means of these enzymes it is possible for the first time to produce silica nanostructures biocatalytically, which opens new ways for construction of biosensors. The cDNAs encoding the responsible enzymes have been isolated and the proteins can be produced in a recombinant way. Here we demonstrate the silicatein-mediated biosilica format…
Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single-molecule three-color FRET.
2021
Cellular function depends on the correct folding of proteins inside the cell. Heat-shock proteins 70 (Hsp70s), being among the first molecular chaperones binding to nascently translated proteins, aid in protein folding and transport. They undergo large, coordinated intra- and interdomain structural rearrangements mediated by allosteric interactions. Here, we applied a three-color single-molecule Forster resonance energy transfer (FRET) combined with three-color photon distribution analysis to compare the conformational cycle of the Hsp70 chaperones DnaK, Ssc1, and BiP. By capturing three distances simultaneously, we can identify coordinated structural changes during the functional cycle. Be…
Citrate Sensing by the C 4 -Dicarboxylate/Citrate Sensor Kinase DcuS of Escherichia coli : Binding Site and Conversion of DcuS to a C 4 -Dicarboxylat…
2007
ABSTRACT The histidine protein kinase DcuS of Escherichia coli senses C 4 -dicarboxylates and citrate by a periplasmic domain. The closely related sensor kinase CitA binds citrate, but no C 4 -dicarboxylates, by a homologous periplasmic domain. CitA is known to bind the three carboxylate and the hydroxyl groups of citrate by sites C1, C2, C3, and H. DcuS requires the same sites for C 4 -dicarboxylate sensing, but only C2 and C3 are highly conserved. It is shown here that sensing of citrate by DcuS required the same sites. Binding of citrate to DcuS, therefore, was similar to binding of C 4 -dicarboxylates but different from that of citrate binding in CitA. DcuS could be converted to a C 4 -…
Antimicrobial activity of methylene blue and toluidine blue O covalently bound to a modified silicone polymer surface
2009
Methylene Blue or Toluidine Blue O were covalently bound to an activated silicone polymer by means of an amide condensation reaction. UV-visible absorption spectra confirmed that the dye was surface bound. The new polymers with covalently attached dye display significant bactericidal activity against Escherichia coli and Staphylococcus epidermidis with a 99.999% reduction in viable bacteria after four minutes exposure to a low power laser.
The Aerobic and Anaerobic Respiratory Chain of Escherichia coli and Salmonella enterica: Enzymes and Energetics.
2014
Escherichia coli contains a versatile respiratory chain that oxidizes 10 different electron donor substrates and transfers the electrons to terminal reductases or oxidases for the reduction of six different electron acceptors. Salmonella is able to use two more electron acceptors. The variation is further increased by the presence of isoenzymes for some substrates. A large number of respiratory pathways can be established by combining different electron donors and acceptors. The respiratory dehydrogenases use quinones as the electron acceptors that are oxidized by the terminal reductase and oxidases. The enzymes vary largely with respect to their composition, architecture, membrane topolog…