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Dobslaff K.,University of Leipzig | Zscharnack K.,University of Leipzig | Kreisig T.,University of Leipzig | Zuchner T.,University of Leipzig | Zuchner T.,Octapharma Biopharmaceuticals GmbH
Amino Acids | Year: 2016

Immunoassays play an essential role in current research and diagnostics resulting in a variety of detection principles. Thereby, homogeneous assays are often used for a fast signal response as demanded for example in point-of-care diagnostics. These systems often rely on a competitive assay design where the sample analyte and the corresponding dye-labeled substance are competing for binding sites on an antibody present in limited amounts. Due to the similar affinities of the antibody towards the sample analyte and the competitor, both sensitivity and assay time are limited. As a consequence, a competitor with a slightly reduced affinity towards the antibody can potentially overcome these drawbacks. Here, we present the rational design of a low-affinity peptide (donor peptide) as a specific analyte competitor for a FRET-based homogeneous immunoassay for the analysis of the protein cystatin C. Thereby, the strategy of peptide-induced antibody generation was combined with the selective variation of the immunization sequence in order to achieve a lower affinity towards the antibody. We could show that shortened donor peptides improved the resulting quenching efficiency in the immunoassay. In addition, the substitution of small hydrophobic amino acids by those with a higher steric demand appeared to be the most promising strategy providing a fast assay response for cystatin C of only 90 s. © 2015 Springer-Verlag Wien.

Rosenlocher J.,University of Applied Sciences, Berlin | Rosenlocher J.,Free University of Berlin | Sandig G.,University of Applied Sciences, Berlin | Kannicht C.,Octapharma Biopharmaceuticals GmbH | And 3 more authors.
Journal of Proteomics | Year: 2016

Glycosylation is the most complex post-translational modification. Thus, it contributes to versatile chemical compositions of proteins, leading to high amounts of protein species. The structural heterogeneity of glycoproteins was also described by the definition of glycoforms. We therefore introduced a new term called "glycoprotein species" to join the two concepts from different fields of biology. In this study, we further determined the theoretical numbers of glycoprotein species of two recombinant glycoproteins - a therapeutical antibody and the human protease inhibitor alpha-1-antitrypsin (A1AT) - based on structural analysis of their N-glycans. Moreover, we showed that variations in the used cell lines and their cultivation conditions strongly influence the number of glycoprotein species in case of recombinant A1AT production. Biological significance: Protein glycosylation is a major source for the huge amount of protein species. This study extends the sight of protein species by the following contributions: 1) The new term "glycoprotein species" was defined to introduce the concept of glycoforms into the field. 2) An estimation of the number of potential glycoprotein species of two particular glycoproteins was given. 3) The influence of production conditions for recombinant glycoproteins on glycoprotein species generation was displayed. © 2015 Elsevier B.V.

Hall V.J.,Copenhagen University | Lindblad M.M.,Copenhagen University | Jakobsen J.E.,University of Aarhus | Gunnarsson A.,University of Aarhus | And 5 more authors.
DMM Disease Models and Mechanisms | Year: 2015

Animal models of familial juvenile onset of Alzheimer's disease (AD) often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw). We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs) isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs) from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased a-and β-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model of the disease. Finally, it might be possible to use large mammal models to model familial AD by insertion of only a single mutation. © 2015. Published by The Company of Biologists Ltd.

Hlavica P.,Walther Straub Institute For Pharmakologie Und Toxikologie Der Lmu | Lehnerer M.,Octapharma Biopharmaceuticals GmbH
Current Drug Metabolism | Year: 2010

In view of the pivotal role played by the diversity of fatty acid-derived oxy-products in a vast array of physiological processes, precise knowledge about the molecular principles dictating substrate specificity and regioselectivity in P450-catalyzed oxidative attack on the distinctly structured carbon chains of the monocarboxylic acids is of paramount importance. Based on a general, CYP102A1-related construct, the majority of prospective key determinants participating in fatty acid recognition/binding were found to cluster near the distal heme face made up by the helical B', F, G and I tetrad as well, as the B'-C interhelical loop and certain β-sheet segments. Most of the contact sites examined show a frequency of conservation <10%, hinting at the requirement of some degree of conformational flexibility. Some decisive elements may also have a function in maintaining active-site integrity, governing substrate access to the catalytic centre, and steering the redox machinery to efficiently promote fatty acid oxidations. Physico-chemical factors imposing constraints on orientation of the fatty acid molecules towards the iron-oxene core focus on the variably expressed polarity profile of the diverse docking regions and bulkiness of critical amino acid side chains, acting as selectivity filters for the substrate homologues. Also, dynamic fluctuations of certain contact sites located in the distal backbone of P450s may impact fatty acid positioning. Genetic engineering to introduce versatile properties into fatty acid hydroxylases may give an impetus to biotechnological exploitation of the tailored enzymes in. the production of fine chemicals and therapeutic agents. © 2010 Bentham Science Publishers Ltd.

Franke S.,University of Leipzig | Kreisig T.,University of Leipzig | Buettner K.,University of Leipzig | Zuchner T.,University of Leipzig | Zuchner T.,Octapharma Biopharmaceuticals GmbH
Analytical and Bioanalytical Chemistry | Year: 2015

As one of the most commonly used enzyme in molecular biology, the T4 DNA ligase presents an important tool for the manipulation of DNA. T4 DNA ligase activity measurements are based on the use of radioactivity or rather labor-intense procedures including gel-based analysis. We therefore established a homogeneous T4 DNA ligase assay utilizing a specifically designed fluorescein- and dark quencher-labeled DNA molecule. Upon ligation of both DNA molecules, a quenching occurs and the fluorescence intensity decreases with increasing ligase concentrations. The assay allows a sensitive and precise quantification (CV, 4.6-5.5 %) of T4 DNA ligase activities and showed a high specificity when tested against other ligases of related and different species. Most importantly, this T4 DNA ligase assay requires only one working and incubation step before measurement can take place at room temperature and may therefore offer an interesting alternative to existing, more laborious ligase assays. © 2014 Springer-Verlag.

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