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K Bayer

Publications and source records attributed to K Bayer.

13 recordsLinked to original sources
PubMed2005

Upper limits on a stochastic background of gravitational waves.

The Laser Interferometer Gravitational-Wave Observatory has performed a third science run with much improved sensitivities of all three interferometers. We present an analysis of approximately 200 hours of data acquired during this run, used to search for a stochastic background of gravitational radiation. We place upper bounds on the energy density stored as gravitational radiation for three different spectral power laws. For the flat spectrum, our limit of omega0 < 8.4 x 10(-4) in the 69-156 Hz band is approximately 10(5) times lower than the previous result in this frequency range.

Journal Article↗
PubMed2005

Limits on gravitational-wave emission from selected pulsars using LIGO data.

We place direct upper limits on the amplitude of gravitational waves from 28 isolated radio pulsars by a coherent multidetector analysis of the data collected during the second science run of the LIGO interferometric detectors. These are the first direct upper limits for 26 of the 28 pulsars. We use coordinated radio observations for the first time to build radio-guided phase templates for the expected gravitational-wave signals. The unprecedented sensitivity of the detectors allows us to set strain upper limits as low as a few times 10(-24). These strain limits translate into limits on the equatorial ellipticities of the pulsars, which are smaller than 10(-5) for the four closest pulsars.

Journal Article↗
PubMed2000

Ferricyanide reduction by Escherichia coli: kinetics, mechanism, and application to the optimization of recombinant fermentations.

Ferricyanide reduction was studied by flow injection analysis (FIA) and chronoamperometry (CA) using two host strains and one recombinant strain of E. coli. Samples taken from batch cultures of E. coli JM105 and HB101 showed maximal specific ferricyanide reduction rates in the late exponential phase of growth, with values (micromol/min x g) of 24 (FIA) and 17 (CA) for JM105, and 36 (FIA) for HB101, when shake-flask cultures were sampled, and 70 for HB101, when a chemostat was used to control pH and dissolved oxygen concentration throughout the cultivation. Remarkably higher ferricyanide reduction rates were obtained with HB101 cells cultivated continuously at very slow growth rate, when chilled, resuspended cell samples were incubated for 5 min in solutions containing 10 mM succinate or formate. These compounds are substrates for primary, membrane-bound dehydrogenases that transfer electrons via ubiquinone to the cytochrome oxidase complexes. Apparent Michaelis-Menten kinetics were observed with respect to ferricyanide concentration when 10 mM succinate was included in the assay buffer; apparent Km values of 10.1+/-0.6 mM and 14.4+/-1.2 mM ferricyanide were obtained for exponential- and stationary-phase E. coli JM105, respectively. Cyanide inhibition studies show that ferricyanide is reduced mainly by cytochrome o oxidase in exponentially growing cells. The large difference in ferricyanide reduction rates observed in the absence and presence of succinate and formate were used to signal stationary-phase entry 5 h after induction of recombinant human Cu/Zn superoxide dismutase expression in a batch fermentation of E. coli HMS174(DE3)(pET3ahSOD). This new method can be used as an adjunct to the quantitation of medium components for the optimization of recombinant fermentations.

Escherichia coli↗
PubMed1999

Metabolic approaches for the optimisation of recombinant fermentation processes.

The aim of this work was the establishment of a novel method to determine the metabolic load on host-cell metabolism resulting from recombinant protein production in Escherichia coli. This tool can be used to develop strategies to optimise recombinant fermentation processes through adjustment of recombinant-protein expression to the biosynthetic capacity of the host-cell. The signal molecule of the stringent-response network, guanosine tetraphosphate (ppGpp), and its precursor nucleotides were selected for the estimation of the metabolic load relating to recombinant-protein production. An improved analytical method for the quantification of nucleotides by ion-pair, high-performance liquid chromatography was established. The host-cell response upon overexpression of recombinant protein in fed-batch fermentations was investigated using the production of human superoxide dismutase (rhSOD) as a model system. E. coli strains with different recombinant systems (the T7 and pKK promoter system) exerting different loads on host-cell metabolism were analysed with regard to intracellular nucleotide concentration, rate of product formation and plasmid copy number.

Chromatography, High Pressure Liquid↗
PubMed1998

Flow cytometric analysis of bacterial physiology during induction of foreign protein synthesis in recombinant Escherichia coli cells.

The production of foreign proteins at high yields represents a severe metabolic stress for Escherichia coli cells. In many cases, induction of protein synthesis results in rapid exhaustion of the cellular energy and metabolic precursors and thus in cell death. Therefore, sustained production of foreign proteins requires some fine tuning of the specific production rate to meet the capabilities of the cell. This has stimulated us to analyze by flow cytometry the physiological behaviour of recombinant E. coli cells producing human superoxide dismutase (SOD). Two strains that produce SOD under the control of either a combined T7/lac promoter or the phi10 promoter were compared by using the following parameters: (a) total DNA content as an indicator of cell division, (b) total RNA content as a measure for protein synthesis activity, (c) total protein content representing cell size, and (d) intracellular SOD content as a measure for productivity. Results show that those cells that continue to increase their biomass after induction of foreign protein synthesis also have the highest specific production rate. Cells, however, do not divide to a measureable degree but rather increase their size. The results confirm the importance of fine-tuning expression systems to prolong the lifetime of cells after induction. This will result in an increased yield.

Bacterial Proteins↗
PubMed1998

Off-line quantitative monitoring of plasmid copy number in bacterial fermentation by capillary electrophoresis.

Capillary electrophoresis (CE) is an effective instrumental alternative to conventional slab gel electrophoresis in the determination of plasmid copy number during recombinant protein formation processes. This analytical setup provides efficient separation of different species of linearized plasmid molecules and quantification by UV detection. Both fused silica and gel-filled capillaries are assessed with respect to peak resolution and reproducibility. The application of coated capillaries eliminates the electroosmatic flow to a large extent, resulting in excellent separation of DNA fragments. The application of UV detection enables the analysis of linearized plasmid DNA with a conventional laboratory CE device. All investigated plasmids show good peak resolution due to their significant differences in molecular size, which is essential for sufficient separation of individual DNA molecules.

Bacteria↗
PubMed1998

Recombinant expression of alliin lyase from garlic (Allium sativum) in bacteria and yeasts.

Recombinant garlic alliin lyase was produced in Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. A cDNA clone was obtained from garlic bulbs by PCR and introduced into suitable bacterial and yeast expression vectors. The recombinant alliin lyase forms inclusion bodies in all three host organisms, which are deposited in the cytoplasm. After cell lysis and harvesting by centrifugation, the inclusion bodies were solubilized in Zwittergent 3-14 solution and refolded by stepwise dilution. Specific alliin lyase activity could be recovered by this procedure.

Blotting, Western↗
PubMed1996

Optimization of recombinant gene expression in Escherichia coli.

The major targets for improvement of recombinant expression efficiency in Escherichia coli are gene dosage, transcription and, to some extent, translation. In order to evaluate the relative importance of these factors, the kinetics of specific mRNA compared to product formation was studied for different widely used expression systems, producing recombinant human superoxide dismutase. For a system employing phage T7 RNA polymerase, where a high level of recombinant protein expression puts a high metabolic burden on the cells, it was shown that transcription is not the limiting factor. To improve the translation rate of a common vector based on the tac promoter, the Shine-Dalgarno (SD) sequence was mutated towards stronger homology to the anti-SD sequence of the E. coli 16S rRNA. A 12.2-fold increase in protein yield was accompanied by a 4.3-fold increase in specific mRNA, indicating that transcription of the recombinant gene is coupled to translation. As this coupling amplifies the detrimental effect of a low-efficiency ribosomal binding site, much attention should be paid to translation initiation when optimizing a recombinant protein production system. Finally, reasons for the high expression level before induction are discussed, and first results towards reducing it are presented.

Base Sequence↗
PubMedDate not supplied

Rational design of an improved induction scheme for recombinant Escherichia coli.

In Escherichia coli, strong overexpression of a recombinant protein has been shown to be deleterious due to a heavy metabolic burden on the host cell, which may completely cease cell growth before maximum product accumulation has occurred. Aiming at a reduction of very high product formation rates, we engineered E. coli strains by mutating the Leloir pathway for galactose metabolization, so that galactose can be utilized to induce lac derived promoters. The induction with galactose was effective in every strain and expression construct tested, and it reduced the metabolic burden on a highly overproducing clone so that cell growth and product accumulation could be maintained for several generations.

Escherichia coli↗
PubMedDate not supplied

America the beautiful?

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Environmental Pollution↗