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Jena, Germany

Biermann M.,Leibniz Institute for Natural Product Research and Infection Biology | Bardl B.,Leibniz Institute for Natural Product Research and Infection Biology | Vollstadt S.,Leibniz Institute for Natural Product Research and Infection Biology | Linnemann J.,Leibniz Institute for Natural Product Research and Infection Biology | And 3 more authors.
Amino Acids | Year: 2013

In this study, a precise and reliable ultra-high performance liquid chromatography (UHPLC) method for the simultaneous determination of non-canonical (norvaline and norleucine) and standard amino acids (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, valine, phenylalanine, isoleucine, leucine) in biopharmaceutical- related fermentation processes was established. After pre-column derivatization with ortho-phthaldialdehyde and 2-mercaptoethanol, the derivatives were separated on a sub-2 μm particle C18 reverse-phase column. Identification and quantification of amino acids were carried out by fluorescence detection. To test method feasibility on standard HPLC instruments, the assay was properly transferred to a core-shell particle C18 reverse-phase column. The limits of detection showed excellent sensitivity by values from 0.06 to 0.17 pmol per injection and limits of quantification between 0.19 and 0.89 pmol. In the present study, the newly established UHPLC method was applied to a recombinant antibody Escherichia coli fermentation process for the analysis of total free amino acids. We were able to specifically detect and quantify the unfavorable amino acids in such complex samples. Since we observed trace amounts of norvaline and norleucine during all fermentation phases, an obligatory process monitoring should be considered to improve quality of recombinant protein drugs in future. © 2013 The Author(s). Source


Ludecke C.,Leibniz Institute for Natural Product Research and Infection Biology | Ludecke C.,Friedrich - Schiller University of Jena | Jandt K.D.,Friedrich - Schiller University of Jena | Siegismund D.,Friedrich - Schiller University of Jena | And 6 more authors.
PLoS ONE | Year: 2014

Biomaterials-associated infections are primarily initiated by the adhesion of microorganisms on the biomaterial surfaces and subsequent biofilm formation. Understanding the fundamental microbial adhesion mechanisms and biofilm development is crucial for developing strategies to prevent such infections. Suitable in vitro systems for biofilm cultivation and bacterial adhesion at controllable, constant and reproducible conditions are indispensable. This study aimed (i) to modify the previously described constant-depth film fermenter for the reproducible cultivation of biofilms at non-depth-restricted, constant and low shear conditions and (ii) to use this system to elucidate bacterial adhesion kinetics on different biomaterials, focusing on biomaterials surface nanoroughness and hydrophobicity. Chemostat-grown Escherichia coli were used for biofilm cultivation on titanium oxide and investigating bacterial adhesion over time on titanium oxide, poly(styrene), poly(tetrafluoroethylene) and glass. Using chemostat-grown microbial cells (single-species continuous culture) minimized variations between the biofilms cultivated during different experimental runs. Bacterial adhesion on biomaterials comprised an initial lag-phase I followed by a fast adhesion phase II and a phase of saturation III. With increasing biomaterials surface nanoroughness and increasing hydrophobicity, adhesion rates increased during phases I and II. The influence of materials surface hydrophobicity seemed to exceed that of nanoroughness during the lag-phase I, whereas it was vice versa during adhesion phase II. This study introduces the non-constant-depth film fermenter in combination with a chemostat culture to allow for a controlled approach to reproducibly cultivate biofilms and to investigate bacterial adhesion kinetics at constant and low shear conditions. The findings will support developing and adequate testing of biomaterials surface modifications eventually preventing biomaterial-associated infections. © 2014 Lüdecke et al. Source


Biermann M.,Leibniz Institute for Natural Product Research and Infection Biology | Linnemann J.,Leibniz Institute for Natural Product Research and Infection Biology | Knupfer U.,Leibniz Institute for Natural Product Research and Infection Biology | Vollstadt S.,Leibniz Institute for Natural Product Research and Infection Biology | And 3 more authors.
Microbial Cell Factories | Year: 2013

Background: Norleucine and norvaline belong to a group of non-canonical amino acids which are synthesized as byproducts in the branched chain amino acid metabolism of Escherichia coli. The earlier observed misincorporation of these rare amino acids into recombinant proteins has attracted increasing attention due to the rising use of protein based biopharmaceuticals in clinical application. Experimental data revealed pyruvate overflow inducing conditions, which typically occur in oxygen limited zones of large-scale fermentations as a major reason leading to norvaline and norleucine synthesis during E. coli cultivation. Previous approaches to suppress misincorporation of norleucine and norvaline considered growth media supplementation with the relevant canonical isostructural compounds, but no research was performed on the impact of the overflow metabolism related trace elements molybdenum, nickel and selenium. These elements form essential parts of the formate hydrogen lyase (FHL) metalloprotein complex, which is a key enzyme of anaerobic pyruvate metabolism in E. coli and could therefore represent a crucial connection to the pyruvate accumulation associated biosynthesis of rare amino acids.Results: In this study, the trace element associated response of recombinant antibody producing E. coli to oxygen limitation at high glucose concentration with a special focus on non-canonical amino acids was analysed. During fed-batch cultivation with provoked oxygen limitation and glucose excess norleucine and norvaline were only accumulated in the absence of molybdenum, nickel and selenium. In contrast, the trace element supplemented stress fermentation showed significantly reduced concentrations of these rare amino acids and the major signature fermentation product formate, supporting the correlation between a functional formate hydrogen lyase complex and low unspecific amino acid synthesis under oxygen limitation at high glucose concentration.Conclusions: The formation of norleucine and norvaline by recombinant E. coli during cultivation with provoked oxygen limitation and glucose excess can be reduced to levels at the detection limit by adding the trace elements molybdenum, selenium and nickel to the fermentation medium. Even under the metabolic burden during induction phase the physiologically available concentrations of non-canonical amino acids remained low. Since our results allow facile process changes that can be easily implemented to avoid the undesirable accumulation of norleucine and norvaline, we consider this study highly interesting for improved process development in E. coli based recombinant drug production and the future development of possible mechanisms to reduce misincorporation events into protein based biopharmaceuticals. © 2013 Biermann et al.; licensee BioMed Central Ltd. Source


Roversi P.,University of Oxford | Ryffel B.,University of Orleans | Ryffel B.,University of Cape Town | Togbe D.,University of Orleans | And 14 more authors.
Journal of Biological Chemistry | Year: 2013

Molecules that simultaneously inhibit independent or co-dependent proinflammatory pathways may have advantages over conventional monotherapeutics.OmCIis a bifunctional protein derived from blood-feeding ticks that specifically prevents complement (C)-mediated C5 activation and also sequesters leukotriene B4 (LTB4) within an internal binding pocket. Here, we examined the effect of LTB4 binding on OmCI structure and function and investigated the relative importance of C-mediated C5 activation and LTB4 in a mouse model of immune complex-induced acute lung injury (IC-ALI). We describe two crystal structures of bacterially expressed OmCI: one binding a C16 fatty acid and the other binding LTB4 (C20). We show that the C5 and LTB4 binding activities of the molecule are independent of each other and that OmCI is a potent inhibitor of experimental IC-ALI, equally dependent on both C5 inhibition and LTB4 binding for full activity. The data highlight the importance of LTB4 in IC-ALI and activation of C5 by the complement pathway C5 convertase rather than by non-C proteases. The findings suggest that dual inhibition of C5 and LTB 4 may be useful for treatment of human immune complex-dependent diseases. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Source


Huang M.,Max Planck Institute for Chemical Ecology | Huang M.,Virginia Polytechnic Institute and State University | Abel C.,Max Planck Institute for Chemical Ecology | Abel C.,BBT Biotech GmbH | And 8 more authors.
Plant Physiology | Year: 2010

When attacked by insects, plants release mixtures of volatile compounds that are beneficial for direct or indirect defense. Natural variation of volatile emissions frequently occurs between and within plant species, but knowledge of the underlying molecular mechanisms is limited. We investigated intraspecific differences of volatile emissions induced from rosette leaves of 27 accessions of Arabidopsis (Arabidopsis thaliana) upon treatment with coronalon, a jasmonate mimic eliciting responses similar to those caused by insect feeding. Quantitative variation was found for the emission of the monoterpene (E)-β-ocimene, the sesquiterpene (E,E)-α-farnesene, the irregular homoterpene 4,8,12-trimethyltridecatetra-1,3,7,11-ene, and the benzenoid compound methyl salicylate. Differences in the relative emissions of (E)-β-ocimene and (E,E)-α-farnesene from accession Wassilewskija (Ws), a high-(E)-β-ocimene emitter, and accession Columbia (Col-0), a trace-(E)-β-ocimene emitter, were attributed to allelic variation of two closely related, tandem-duplicated terpene synthase genes, TPS02 and TPS03. The Ws genome contains a functional allele of TPS02 but not of TPS03, while the opposite is the case for Col-0. Recombinant proteins of the functional Ws TPS02 and Col-0 TPS03 genes both showed (E)-β-ocimene and (E,E)-α-farnesene synthase activities. However, differential subcellular compartmentalization of the two enzymes in plastids and the cytosol was found to be responsible for the ecotype-specific differences in (E)-β-ocimene/(E,E)-α-farnesene emission. Expression of the functional TPS02 and TPS03 alleles is induced in leaves by elicitor and insect treatment and occurs constitutively in floral tissues. Our studies show that both pseudogenization in the TPS family and subcellular segregation of functional TPS enzymes control the variation and plasticity of induced volatile emissions in wild plant species. © 2010 American Society of Plant Biologists. Source

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