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Department of Cell Biology and Applied Virology
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Department of Biophysics and Cryotechnology
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    ABSTRACT: The enzyme horseradish peroxidase has been immobilized on nanoelectrode arrays by alternating current dielectrophoresis. Preservation of its enzymatic function after field application was demonstrated by oxidizing dihydrorhodamine 123 with hydrogen peroxide as co-oxidant to create its fluorescent form, rhodamine 123. Localization of the fluorescently labeled enzyme and its product was conducted by fluorescence microscopy. Nanoelectrodes were prepared as tungsten pins arranged in square arrays. Experimental parameters for dielectrophoretic immobilization were optimized for even enzyme distribution and for enzymatic efficiency. Enzyme activity was quantified by determination of fluorescence intensities of immobilized enzyme molecules and of rhodamine 123 produced. These results demonstrate that DEP can be applied to immobilize enzyme molecules while retaining their activity and rendering any chemical modifications unnecessary. This introduces a novel way for the preparation of bioactive surfaces for processes such as biosensing.
    Electrophoresis 02/2014; 35(4). DOI:10.1002/elps.201300447
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    ABSTRACT: Protein expression systems are widely used in biotechnology and medicine for the efficient and economic production of therapeutic proteins. Today, cultivated Chinese hamster ovary (CHO) cells are the market dominating mammalian cell-line for the production of complex therapeutic proteins. Despite this outstanding potential of CHO cells, no high-yield cell-free system based on translationally active lysates from these cells has been reported so far. To date, CHO cell extracts have only been used as a foundational research tool for understanding mRNA translation (Lodish et al., 1974; McDowell et al., 1972). In the present study, we address this fact by establishing a novel cell-free protein expression system based on extracts from cultured CHO cells. Lysate preparation, adaptation of in vitro reaction conditions and the construction of particular expression vectors are considered for high-yield protein production. A specific in vitro expression vector, which includes an internal ribosome entry site (IRES) from the intergenic region (IGR) of the Cricket paralysis virus (CrPV), has been constructed in order to obtain optimal performance. The IGR IRES is supposed to bind directly to the eukaryotic 40S ribosomal subunit thereby bypassing the process of translation initiation, which is often a major bottleneck in cell-free systems. The combination of expression vector and optimized CHO cell extracts enables the production of approximately 50 µg/mL active firefly luciferase within 4 h. The batch-type cell-free coupled transcription-translation system has the potential to perform post-translational modifications, as shown by the glycosylation of erythropoietin. Accordingly, the system contains translocationally active endogenous microsomes, enabling the co-translational incorporation of membrane proteins into biological membranes. Hence, the presented in vitro translation system is a powerful tool for the fast and convenient optimization of expression constructs, the specific labeling of integral membrane proteins and the cell-free production of posttranslationally modified proteins. Biotechnol. Bioeng. 2013;9999: 1-12. © 2013 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 01/2014; 111(1). DOI:10.1002/bit.25013
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    ABSTRACT: Cultivation and proliferation of stem cells in three-dimensional (3-D) scaffolds is a promising strategy for regenerative medicine. Mesenchymal stem cells with their potential to differentiate in various cell types, cryopreserved adhesion-based in fabricated scaffolds of biocompatible materials can serve as ready-to-use transplantation units for tissue repair, where pores allow a direct contact of graft cells and recipient tissue without further preparation. A successful cryopreservation of adherent cells depends on attachment and spreading processes that start directly after cell seeding. Here, we analyzed different cultivation times (0.5, 2, 24 h) prior to adhesion-based cryopreservation of human mesenchymal stem cells within alginate-gelatin cryogel scaffolds and its influence on cell viability, recovery and functionality at recovery times (0, 24, 48 h) in comparison to non-frozen control. Analysis with confocal laser scanning microscopy and scanning electron microscopy indicated that 2 h cultivation time enhanced cryopreservation success: cell number, visual cell contacts, membrane integrity, motility, as well as spreading were comparable to control. In contrast, cell number by short cultivation time (0.5 h) reduced dramatically after thawing and expanded cultivation time (24 h) decreased cell viability. Our results provide necessary information to enhance the production and to store ready-to-use transplantation units for application in bone, cartilage or skin regenerative therapy.
    Journal of Materials Science Materials in Medicine 12/2013; 25(3). DOI:10.1007/s10856-013-5108-x


  • Address
    Ensheimer Straße 48, 66386 St. Ingbert, Sankt Ingbert, Germany
  • Head of Institution
    Prof. Dr. Günter Fuhr
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    +49 (0) 6894 980 - 0
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    +49 (0) 6894 980 - 400
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Top publications last week by reads

Particle and Fibre Toxicology 02/2011; 8(1):8. DOI:10.1186/1743-8977-8-8
23 Reads
Advances in biochemical engineering/biotechnology 11/2013; 140. DOI:10.1007/10_2013_251
8 Reads

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