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Halle-neustadt, Germany

Biologicals are one of the most dynamic growing markets in the field of therapeutics. Biosimilars unlike traditional generic pharmaceuticals copy a complex three dimensional structure based on large molecules. Process and product are closely related to each other. In the case of biosimilars, the production process development is one of the most ambitious aspects during development. The challenge to combine new technologies and economical requirements with a predefined product quality implies a specialized organization to develop these kinds of processes. © Springer-Verlag 2014. Source

Singer A.U.,University of Toronto | Schulze S.,Martin Luther University of Halle Wittenberg | Skarina T.,University of Toronto | Xu X.,University of Toronto | And 10 more authors.
PLoS Pathogens | Year: 2013

Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen Xanthomonas campestris pv. vesicatoria exhibits E3 ubiquitin ligase activity in vitro and in planta, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the Xanthomonas XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity. © 2013 Singer et al. Source

Hoffmann A.,Martin Luther University of Halle Wittenberg | Kovermann M.,Martin Luther University of Halle Wittenberg | Lilie H.,Martin Luther University of Halle Wittenberg | Fiedler M.,Scil Proteins | And 3 more authors.
PLoS ONE | Year: 2012

A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1:3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins - designed ankyrin repeat proteins - without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies. © 2012 Hoffmann et al. Source

Scil Proteins | Date: 2011-04-20

The invention relates to a method for producing folded prethrombin, wherein inclusion bodies, which contain unfolded prethrombin or a derivative thereof, are solubilized in a solubilization buffer containing at least one chaotropic compound and at least one organic disulfide compound. The invention further relates to methods for producing thrombin and a-thrombin and derivatives thereof. The invention also relates to solutions that contain folded proteins, which can be produced by the methods according to the invention.

The present invention concerns a protein mixture comprising at least a first fusion protein comprising a protein or protein fragment, and an interaction domain and a protein translocation sequence, which effects that the fusion protein upon expression in a bacterium is translocated through the cytoplasmic membrane in an essentially unfolded state and at least a second fusion protein comprising a protein or protein fragment, and an interaction domain and a protein translocation sequence which effects that the fusion protein is translocated through the cytoplasmic membrane upon expression in a bacterium in an essentially folded state, wherein the interaction domain of the first protein can bind to those of the second protein.

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