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1دورية أكاديمية
المؤلفون: Andreas Hoffmann, Michael Kovermann, Hauke Lilie, Markus Fiedler, Jochen Balbach, Rainer Rudolph, Sven Pfeifer
المصدر: PLoS ONE, Vol 7, Iss 2, p e31298 (2012)
الوصف: 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.
وصف الملف: electronic resource
العلاقة: http://europepmc.org/articles/PMC3282696?pdf=renderTest; https://doaj.org/toc/1932-6203Test
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2دورية أكاديمية
المؤلفون: Edith S. A. Hofinger, Martin Spickenreither, Jan Oschmann, Rainer Rudolph, Armin Buschauer
المساهمون: The Pennsylvania State University CiteSeerX Archives
مصطلحات موضوعية: lase
الوصف: The human hyaluronidase Hyal-1, one of six human hya-luronidase subtypes, preferentially degrades hyaluronic acid present in the extracellular matrix of somatic tissues. Modulations of Hyal-1 expression have been observed in a number of malignant tumors. However, its role in disease progression is discussed controversially due to limited information on enzyme properties as well as the lack of specific inhibitors. Therefore, we expressed human Hyal-1 in a prokaryotic and in an insect cell system to produce larger amounts of the purified enzyme. In Escherichia coli, Hyal-1 formed inclusion bodies and was refolded in vitro after purification by metal ion affinity chromatography. However, the enzyme was produced with extremely low folding yields (0.5%) and exhibited a low specific activity (0.1 U/mg). Alternatively, Hyal-1 was secreted into the medium of stably transfected Drosophila Schneider-2 (DS-2) cells. After several purification steps, highly pure enzyme with a specific activity of 8.6 U/mg (consistent with the reported activity of human Hyal-1 from plasma) was obtained. Both Hyal-1 enzymes showed pH profiles similar to the hyaluronidase of human plasma with an activity maximum at pH 3.5–4.0. Deglycosylation of Hyal-1, expressed in DS-2 cells, resulted in a decrease in the enzymatic activity determined by a colorimetric hyaluro-nidase activity assay. Purified Hyal-1 from DS-2 cells was used for the investigation of the inhibitory activity of new ascorbic acid derivatives. Within this series, L-ascorbic acid tridecanoate was identified as the most potent inhibitor with an IC50 of 50+ 4 mM comparable with glycyrrhizic acid. Key words: Drosophila Schneider-2 cells/Hyal-1/ hyaluronidase/inhibitors/protein folding
وصف الملف: application/pdf
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3دورية أكاديمية
المؤلفون: Ajamaluddin Malik, Rainer Rudolph, Brigitte Söhling
المساهمون: The Pennsylvania State University CiteSeerX Archives
الوصف: production of native human pepsinogen in the bacterial periplasm Protein Expr. Purif.
وصف الملف: application/pdf
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4
المؤلفون: Eva Bosse-Doenecke, Rainer Rudolph, Mohanraj Gopalswamy, Jochen Balbach, Nils Drechsler, Günther Jahreis, Julia Fröbel
المصدر: Biophysical Chemistry. 154:66-72
مصطلحات موضوعية: chemistry.chemical_classification, Magnetic Resonance Spectroscopy, Chemistry, Parathyroid hormone receptor, Molecular Sequence Data, Organic Chemistry, Biophysics, Parathyroid hormone, Isothermal titration calorimetry, Peptide, Peptide binding, Calorimetry, Biochemistry, Recombinant Proteins, Protein Structure, Tertiary, Ectodomain, Humans, Amino Acid Sequence, Receptor, hormones, hormone substitutes, and hormone antagonists, Protein Binding, Receptor, Parathyroid Hormone, Type 1, G protein-coupled receptor
الوصف: The parathyroid hormone (PTH)1 receptor is a member of the class B G protein-coupled receptor (GPCR) family and regulates bone and mineral metabolism of vertebrates. A truncated highly active parathyroid hormone fragment PTH (1–34) exerts stimulatory effects on the receptor and is used for treatment of osteoporosis. To study the interacting amino acids of the natural peptide ligand PTH (1–84) with the ectodomain of its receptor we used peptide micro arrays on solid cellulose membranes. The amino acids Arg20 and Trp23 within the identified core binding stretch PTH (20–26) were found to be most important for affinity to the ectodomain of PTH1R. Isothermal titration calorimetry and NMR spectroscopy allowed peptide binding studies in solution and verified peptide positions required for high affinity. With this combination of biochemical and biophysical methods we extend former findings on this essential interaction and can now provide a strategy to screen for optimized therapeutic peptides.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::f68278294a9013b616a4fe7a1bda2135Test
https://doi.org/10.1016/j.bpc.2011.01.002Test -
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المصدر: Journal of Biotechnology. 150:64-72
مصطلحات موضوعية: Anions, Protein Folding, Ionic Liquids, Bioengineering, Alkylation, Protein aggregation, Applied Microbiology and Biotechnology, Chloride, Plasminogen Activators, chemistry.chemical_compound, medicine, Organic chemistry, Alkyl, chemistry.chemical_classification, Proteinogenic amino acid, Aqueous solution, Calorimetry, Differential Scanning, Protein Stability, Imidazoles, Tryptophan, Proteins, General Medicine, Models, Chemical, Solubility, chemistry, Ionic liquid, Muramidase, Salts, Biotechnology, medicine.drug
الوصف: Several ionic liquids have been shown to act as suppressors of protein aggregation and to effectively promote the in vitro refolding of denatured proteins. In this work, the influence of the anion of these organic salts on their performance as refolding enhancers was tested, using a series of N-ethyl-N'-methyl imidazolium (EMIM) salts. Variation of the anion had a profound effect on the renaturation of the recombinant plasminogen activator rPA, which served as a model protein. The effect was roughly correlated with the stability of proteins in the tested aqueous ionic liquid solutions. Strongly destabilizing anions with higher alkyl substitutions like, e.g., hexyl sulfate were unable to promote protein refolding. However, the dimethyl and diethyl phosphate salts, which are known to be quite compatible with protein stability, also effectively suppressed renaturation, while alkylated sulfate anions with the same influence on protein stability did promote in vitro refolding. Only ionic liquid co-solvent systems with an intermediate capacity for solubilizing the proteinogenic amino acid tryptophan were found to permit effective renaturation of the model protein rPA. The most effective refolding enhancer among the tested ionic liquids was EMIM chloride.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::320408db7b0b41ce40694eed3db70bdbTest
https://doi.org/10.1016/j.jbiotec.2010.07.003Test -
6
المؤلفون: Rainer Rudolph, S. von Einem, Elisabeth Schwarz
المصدر: Protein Expression and Purification. 73:65-69
مصطلحات موضوعية: Inclusion Bodies, Protein Folding, Chemistry, Oxidative folding, Bone Morphogenetic Protein 2, Biological activity, Alkaline Phosphatase, Bone morphogenetic protein, Bone morphogenetic protein 2, Recombinant Proteins, law.invention, Folding (chemistry), Biochemistry, law, Enzyme Induction, Intramolecular force, Escherichia coli, Biophysics, Recombinant DNA, Humans, Electrophoresis, Polyacrylamide Gel, Oxidation-Reduction, Biotechnology, Protein Renaturation
الوصف: Bone morphogenetic proteins (BMPs) stimulate bone formation and thus constitute important protein therapeutics. Here, a novel procedure is presented which allows fast and efficient production of biologically active BMP-2 via a TWO-STEP procedure: the conditions are designed such that the first step favors formation of monomeric species with the correct intramolecular disulfide bridges, the conditions of the second folding reaction stimulate the formation of the intermolecular disulfide bridge. The short processing times and increased yields compared to previously published procedures allow low-cost production of this important protein drug.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::3742a1f73b36c1f210384c26f06a20e5Test
https://doi.org/10.1016/j.pep.2010.03.009Test -
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المؤلفون: Christian Ihling, Rainer Rudolph, Kai Tittmann, Andrea Sinz, Kathrin Schröder-Tittmann, Eva Bosse-Doenecke, Steffen Reedtz-Runge
المصدر: Biochemistry. 49:7956-7965
مصطلحات موضوعية: Agonist, Circular dichroism, medicine.drug_class, Recombinant Fusion Proteins, Ligands, medicine.disease_cause, Biochemistry, Glucagon-Like Peptide-1 Receptor, Inclusion bodies, 03 medical and health sciences, 0302 clinical medicine, Escherichia coli, Receptors, Glucagon, medicine, Humans, Receptor, 030304 developmental biology, 0303 health sciences, biology, Chemistry, Ligand (biochemistry), Cytosol, Chaperone (protein), biology.protein, 030217 neurology & neurosurgery
الوصف: Activation of the glucagon-like peptide-1 receptor (GLP-1R) upon ligand binding leads to the release of insulin from pancreatic cells. This strictly glucose-dependent process renders the receptor and its ligands useful in the treatment of type II diabetes mellitus. To enable a biophysical characterization in vitro, we expressed the human full-length GLP-1R in the cytosol of Escherichia coli as inclusion bodies. After purification, refolding of the SDS-solubilized receptor was achieved by the exchange of SDS against the detergent Brij78 using an artificial chaperone system. Far-UV circular dichroism spectroscopic studies revealed that the receptor adopts a characteristic alpha-helical structure in Brij78 micelles. Ligand binding of the renatured protein was quantified by fluorescence quenching and surface plasmon resonance spectroscopy. In the presence of Brij micelles, the refolded receptor binds the agonist exendin-4 with an apparent dissociation constant of approximately 100 nM in a reversible one-step mechanism. To demonstrate that the detected ligand binding activity is not only due to an autonomously functional N-terminal domain (nGLP-1R) but also due to additional contacts with the juxtamembrane part, we separately expressed and refolded the extracellular domain relying on identical protocols established for the full-length GLP-1R. In support of the suggested multidomain binding mode, the nGLP-1R binds exendin-4 with a lower affinity (K(app) in the micromolar range) and a different kinetic mechanism. The lower ligand affinity of the nGLP-1R results entirely from a decreased kinetic stability of the receptor-ligand complex, dissociation of which is approximately 40-fold faster in the case of the nGLP-1R compared to the full-length GLP-1R. In summary, a framework was developed to produce functional human full-length GLP-1R by recombinant expression in E. coli as a prerequisite for eventual structure determination and a rigorous biophysical characterization including protein variants.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::f33765c37e0da8f87a28c9ebaaca6c82Test
https://doi.org/10.1021/bi101159sTest -
8
المؤلفون: Rainer Rudolph, Alexander Tischer, Hauke Lilie, Christian Lange
المصدر: Protein Science. 19:1783-1795
مصطلحات موضوعية: Circular dichroism, Arginine, Protein aggregation, Biochemistry, chemistry.chemical_compound, Mechanism of action, chemistry, medicine, Protein folding, medicine.symptom, Solubility, Guanidine, Molecular Biology, Plasminogen activator
الوصف: l-Arginine hydrochloride (l-ArgHCl) was found to be an effective enhancer for in vitro protein refolding more than two decades ago. A detailed understanding of the mechanism of action, by which l-ArgHCl as co-solvent is capable to effectively suppress protein aggregation, while protein stability is preserved, has remained elusive. Concepts for the effects of co-solvents, which have been established over the last decades, were found to be insufficient to completely explain the effects of l-ArgHCl on protein refolding. In this article, we present data, which clearly establish that l-ArgHCl acts on the equilibrium solubility of the native model protein recombinant plasminogen activator (rPA), while for S-carboxymethylated rPA (IAA-rPA) that served as a model protein for denatured protein states, equilibrium solubilities could not be obtained. Solid to solute free transfer energies for native rPA were lowered by up to 14 kJ mol-1 under the tested conditions. This finding is in marked contrast to a previously proposed model in which l-ArgHCl acts as a neutral crowder which exclusively has an influence on the stability of the transition state of aggregation. The effects on the apparent solubility of IAA-rPA, as well as on the aggregation kinetics of all studied protein species, that were observed in the present work could tentatively be explained within the framework of a nucleation-aggregation scheme, in which l-ArgHCl exerts a strong effect on the pre-equilibria leading to formation of the aggregation seed.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::1f7fa3c6675df9a59d671cc285f1433eTest
https://doi.org/10.1002/pro.465Test -
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المؤلفون: Marie-Eve Gravière, Rainer Rudolph, Giuliano Sciara, Julie Lichière, Marina Isabella Siponen, Céline Huyghe, Renaud Wagner, Kerstin Michalke, Christian Cambillau, Christine Magg, Aline Desmyter, Isabelle Lepaul
المساهمون: Architecture et fonction des Macromolécules Biologiques - UMR 6098 (AFMB), Université de Provence - Aix-Marseille 1-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU), Centre National de la Recherche Scientifique (CNRS)-Université de Provence - Aix-Marseille 1
المصدر: Analytical Biochemistry
Analytical Biochemistry, 2010, 401 (1), pp.74-80. ⟨10.1016/j.ab.2010.02.017⟩
Analytical Biochemistry, Elsevier Masson, 2010, 401 (1), pp.74-80. ⟨10.1016/j.ab.2010.02.017⟩مصطلحات موضوعية: Protein Folding, [SDV]Life Sciences [q-bio], Biophysics, 010402 general chemistry, 01 natural sciences, Biochemistry, Inclusion bodies, Mice, 03 medical and health sciences, Receptor, Cannabinoid, CB1, Cell surface receptor, Protein purification, Escherichia coli, Animals, Humans, Refolding, 5-HT5A receptor, G protein-coupled receptor, Receptor, Molecular Biology, Receptor, Parathyroid Hormone, Type 1, 030304 developmental biology, Inclusion Bodies, Cyclodextrins, 0303 health sciences, biology, Sciences du Vivant [q-bio]/Biotechnologies, Cell Biology, Recombinant Proteins, 0104 chemical sciences, Hormone receptor, Rhodopsin, biology.protein, Protein expression, Protein Binding
الوصف: G protein-coupled receptors (GPCRs) represent approximately 3% of the human proteome. They are involved in a large number of diverse processes and, therefore, are the most prominent class of pharmacological targets. Besides rhodopsin, X-ray structures of classical GPCRs have only recently been resolved, including the beta1 and beta2 adrenergic receptors and the A2A adenosine receptor. This lag in obtaining GPCR structures is due to several tedious steps that are required before beginning the first crystallization experiments: protein expression, detergent solubilization, purification, and stabilization. With the aim to obtain active membrane receptors for functional and crystallization studies, we recently reported a screen of expression conditions for approximately 100 GPCRs in Escherichia coli, providing large amounts of inclusion bodies, a prerequisite for the subsequent refolding step. Here, we report a novel artificial chaperone-assisted refolding procedure adapted for the GPCR inclusion body refolding, followed by protein purification and characterization. The refolding of two selected targets, the mouse cannabinoid receptor 1 (muCB1R) and the human parathyroid hormone receptor 1 (huPTH1R), was achieved from solubilized receptors using detergent and cyclodextrin as protein folding assistants. We could demonstrate excellent affinity of both refolded and purified receptors for their respective ligands. In conclusion, this study suggests that the procedure described here can be widely used to refold GPCRs expressed as inclusion bodies in E. coli.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::522c2d66690a2c7d7111b990e4322ab5Test
https://doi.org/10.1016/j.ab.2010.02.017Test -
10
المؤلفون: Lotte Bjerre Knudsen, Patrick William Garibay, Sven Hastrup, Steffen Reedtz-Runge, Rainer Rudolph, Günther H.J. Peters, Christina Rye Underwood
المصدر: The Journal of Biological Chemistry
Underwood, C R, Garibay, P, Knudsen, L B, Hastrup, S, Peters, G H J, Rudolph, R & Reedtz-Runge, S 2010, ' Crystal Structure of Glucagon-like Peptide-1 in Complex with the Extracellular Domain of the Glucagon-like Peptide-1 Receptor ', Journal of Biological Chemistry, vol. 285, no. 1, pp. 723-730 . https://doi.org/10.1074/jbc.M109.033829Testمصطلحات موضوعية: Models, Molecular, Agonist, endocrine system, EGF-like domain, medicine.drug_class, Molecular Sequence Data, Biology, Crystallography, X-Ray, Biochemistry, Glucagon-Like Peptide-1 Receptor, Protein Structure, Secondary, Cell Line, Glucagon-Like Peptide 1, Receptors, Glucagon, Extracellular, medicine, Humans, Amino Acid Sequence, Receptor, Molecular Biology, digestive, oral, and skin physiology, Cell Biology, Protein Structure, Tertiary, Solutions, Competitive antagonist, Protein Structure and Folding, Mutagenesis, Site-Directed, Biophysics, Mutant Proteins, MATH domain, Extracellular Space, hormones, hormone substitutes, and hormone antagonists, Endogenous agonist, Protein Binding, Binding domain
الوصف: GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic beta-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9-39). Interestingly, the isolated extra-cellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 angstrom resolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist-and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous alpha-helix from Thr(13) to Val(33) when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor.
وصف الملف: application/pdf
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::3f3f946707bfa52684465bc418c99567Test
https://doi.org/10.1074/jbc.m109.033829Test
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