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In JoVE (1)
- Klinisch onderzoek en het ruggenmerg verwijderen in een muismodel voor Amyotrofische Lateraal Sclerose (ALS)
Other Publications (4)
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Articles by Martin Suhr in JoVE
Klinisch onderzoek en het ruggenmerg verwijderen in een muismodel voor Amyotrofische Lateraal Sclerose (ALS)
René Günther1, Martin Suhr1, Jan C. Koch1, Mathias Bähr1,2, Paul Lingor1,2, Lars Tönges1
1Dept. of Neurology, University Medicine Göttingen, 2DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany
Een muismodel voor amyotrofische lateraal sclerose (ALS) is klinisch en gedragsmatig onderzocht. Als voorwaarde voor een begeleidende immunohistologische analyse de voorbereiding van het ruggenmerg wordt afgebeeld in detail.
Other articles by Martin Suhr on PubMed
High-yield Expression in Escherichia Coli, Purification, and Characterization of Properly Folded Major Peanut Allergen Ara H 2
Protein Expression and Purification. Oct, 2003 | Pubmed ID: 14550644
Allergic reactions to peanuts are a serious health problem because of their high prevalence, associated with potential severity, and chronicity. One of the three major allergens in peanut, Ara h 2, is a member of the conglutin family of seed storage proteins. Ara h 2 shows high sequence homology to proteins of the 2S albumin family. Presently, only very few structural data from allergenic proteins of this family exist. For a detailed understanding of the molecular mechanisms of food-induced allergies and for the development of therapeutic strategies knowledge of the high-resolution three-dimensional structure of allergenic proteins is essential. We report a method for the efficient large-scale preparation of properly folded Ara h 2 for structural studies and report CD-spectroscopic data. In contrast to other allergenic 2S albumins, Ara h 2 exists as a single continuous polypeptide chain in peanut seeds, and thus heterologous expression in Escherichia coli was possible. Ara h 2 was expressed as Trx-His-tag fusion protein in E. coli Origami (DE3), a modified E. coli strain with oxidizing cytoplasm which allows the formation of disulfide bridges. It could be shown that recombinant Ara h 2, thus overexpressed and purified, and the allergen isolated from peanuts are identical as judged from immunoblotting, analytical HPLC, and circular dichroism spectra.
Development of an Epitope-specific Analytical Tool for the Major Peanut Allergen Ara H 2 Using a High-density Multiple-antigenic Peptide Strategy
Molecular Nutrition & Food Research. Nov, 2004 | Pubmed ID: 15508180
Using the major peanut allergen Ara h 2 as an example, an analytical tool enabling the determination of immunoglobulin E (IgE)-epitopes in processed food allergens was developed. We synthesized a multiple-antigenic peptide (MAP) of the IgE-reactive linear epitope 3 (amino acid positions 27-36) of Ara h 2 and raised a monospecific antiserum against this epitope to obtain a positive control for future epitope resolved diagnostics. First, a MAP of epitope 3, having a molecular mass of 7770 Da, was synthesized, purified, and its structure confirmed by liquid chromatography-mass spectrometry (electrospray ionization) (LC-MS(ESI)), matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), and Edman sequencing. The MAP was then used to raise high titer antibodies in rabbits using the adjuvant Titermax and to characterize the specificity of IgE from allergenic patients sensitized to Ara h 2. The antiserum exclusively detects Ara h 2 in crude peanut extract with a titer of 10(7) by Western blot and reacts specifically with epitope 3 shown by epitope mapping for a library of solid-phase-bound synthetic 15-mer peptides covering the entire sequence of Ara h 2. Such IgE-reactive epitopes are of high analytical relevance as they could constitute the basis for epitope-specific detection systems for use in quality control in the food industry or for forensic purposes in cases of fatal reactions to otherwise undetected peanut proteins.
Isolation and Characterization of Natural Ara H 6: Evidence for a Further Peanut Allergen with Putative Clinical Relevance Based on Resistance to Pepsin Digestion and Heat
Molecular Nutrition & Food Research. Oct, 2004 | Pubmed ID: 15672479
Peanut allergy is a significant health problem because of its prevalence and the potential severity of the allergic reaction. The characterization of peanut allergens is crucial to the understanding of the mechanism of peanut allergy. Recently, we described cloning of the peanut allergen Ara h 6. The aim of this study was isolation and further characterization of nAra h 6. We purified nAra h 6 from crude peanut extract using gel filtration and anion exchange chromatography. The preparation was further characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) with subsequent immunoblotting. Stability of nAra h 6 was studied by an in vitro digestibility assay as well as by resistance against thermal processing. Sequencing of nAra h 6 identified the N-terminal amino acid sequence as MRRERGRQGDSSS. Further results clearly demonstrated stability of nAra h 6 against pepsin digestion and heating. Immunoglobulin G (IgE) binding analysis and its biological activity shown by RBL 25/30-test of natural Ara h 6 supported the importance of this peanut allergen. Investigation of nAra h 6 revealed evidence for a further peanut allergen with putative clinical relevance based on resistance to pepsin digestion and heat.
Structure and Stability of 2S Albumin-type Peanut Allergens: Implications for the Severity of Peanut Allergic Reactions
The Biochemical Journal. May, 2006 | Pubmed ID: 16372900
Resistance to proteolytic enzymes and heat is thought to be a prerequisite property of food allergens. Allergens from peanut (Arachis hypogaea) are the most frequent cause of fatal food allergic reactions. The allergenic 2S albumin Ara h 2 and the homologous minor allergen Ara h 6 were studied at the molecular level with regard to allergenic potency of native and protease-treated allergen. A high-resolution solution structure of the protease-resistant core of Ara h 6 was determined by NMR spectroscopy, and homology modelling was applied to generate an Ara h 2 structure. Ara h 2 appeared to be the more potent allergen, even though the two peanut allergens share substantial cross-reactivity. Both allergens contain cores that are highly resistant to proteolytic digestion and to temperatures of up to 100 degrees C. Even though IgE antibody-binding capacity was reduced by protease treatment, the mediator release from a functional equivalent of a mast cell or basophil, the humanized RBL (rat basophilic leukaemia) cell, demonstrated that this reduction in IgE antibody-binding capacity does not necessarily translate into reduced allergenic potency. Native Ara h 2 and Ara h 6 have virtually identical allergenic potency as compared with the allergens that were treated with digestive enzymes. The folds of the allergenic cores are virtually identical with each other and with the fold of the corresponding regions in the undigested proteins. The extreme immunological stability of the core structures of Ara h 2 and Ara h 6 provides an explanation for the persistence of the allergenic potency even after food processing.