| dc.rights.license | CC0 | en_US |
| dc.contributor.author | Ruth, Nadia | |
| dc.contributor.author | QUINTING, Birgit | |
| dc.contributor.author | Mainil, Jacques | |
| dc.contributor.author | Hallet, Bernard | |
| dc.contributor.author | Frère, Jean-Marie | |
| dc.contributor.author | Huygen, Kris | |
| dc.contributor.author | Galleni, Moreno | |
| dc.date.accessioned | 2021-09-21T07:49:07Z | |
| dc.date.available | 2021-09-21T07:49:07Z | |
| dc.date.issued | 2008 | |
| dc.identifier.issn | 1432-1033 | en_US |
| dc.identifier.uri | https://luck.synhera.be/handle/123456789/1158 | |
| dc.identifier.doi | https://doi.org/10.1111/j.1742-4658.2008.06646.x | en_US |
| dc.description.abstract | 275 (2008), p. 5150-5160 | en_US |
| dc.description.abstractfr | L'insertion de séquences peptidiques hétérologues dans une protéine porteuse peut imposer des contraintes structurelles qui pourraient aider le peptide à adopter un repliement approprié. Ce concept pourrait être le point de départ pour le développement d'une nouvelle génération de vaccins sous-unitaires sûrs basés sur l'expression d'épitopes faiblement immunogènes. Dans la présente étude, nous avons caractérisé la tolérance de la -lactamase de classe TEMA TEM-1 à l'insertion de deux peptides différents, la boucle V3 de la protéine gp120 du VIH, et l'entérotoxine STa thermostable produite par Escherichia coli entérotoxique. L'insertion de la boucle V3 de la protéine gp120 du VIH dans l'échafaudage TEM-1 a donné des protéines insolubles et mal produites. En revanche, quatre β-lactamases hybrides portant le peptide STa ont été efficacement produites et purifiées. L'immunisation de souris BALB/c avec ces protéines hybrides a produit des niveaux élevés d'anticorps spécifiques de TEM-1, ainsi que des niveaux significatifs d'anticorps neutralisants contre STa. | en_US |
| dc.description.abstracten | Insertion of heterologous peptide sequences into a protein carrier may impose structural constraints that could help the peptide to adopt a proper fold. This concept could be the starting point for the development of a new generation of safe subunit vaccines based on the expression of poorly immunogenic epitopes. In the present study, we characterized the tolerance of the TEM-1 class A β-lactamase to the insertion of two different peptides, the V3 loop of the gp120 protein of HIV, and the thermostable STa enterotoxin produced by enterotoxic Escherichia coli. Insertion of the V3 loop of the HIV gp120 protein into the TEM-1 scaffold yielded insoluble and poorly produced proteins. By contrast, four hybrid β-lactamases carrying the STa peptide were efficiently produced and purified. Immunization of BALB/c mice with these hybrid proteins produced high levels of TEM-1-specific antibodies, together with significant levels of neutralizing antibodies against STa. | en_US |
| dc.description.sponsorship | OTH | en_US |
| dc.description.tableofcontents | Results
Insertion of the V3, V3P and STa epitopes at different positions of TEM-1
Production and purification of the hybrid proteins
Enzymatic activity of the hybrid β-lactamases
Enterotoxicity of the TEM–STa hybrid proteins measured by suckling mouse assay
Production of antibodies against the carrier protein TEM-1 and the STa enterotoxin
Neutralization of the STa enterotoxicity
Discussion
Experimental procedures
Antibiotics, chemicals and enzymes
Plasmids, bacterial strains and culture conditions
Construction of synthetic DNA linkers coding for the V3 and STa epitopes
Construction of the hybrid β-lactamases
Measurement of the MIC
Cellular localization of the hybrid β-lactamases
Production and purification of the TEM–STa hybrid proteins
N-terminal sequencing of protein
MS
Determination of kinetic parameters
Suckling mouse assay
Immunization
Measurement of specific IgG antibody production
Antibody neutralization of STa enterotoxicity | en_US |
| dc.language.iso | EN | en_US |
| dc.publisher | Blackwell Science (Oxford) | en_US |
| dc.relation.ispartof | FEBS Journal | en_US |
| dc.rights.uri | ? | en_US |
| dc.subject | class A β-lactamase TEM-1 | en_US |
| dc.subject | hybrid protein | en_US |
| dc.subject | insertion | en_US |
| dc.subject | STa enterotoxin | en_US |
| dc.subject | V3 loop | en_US |
| dc.subject.fr | classe A β-lactamase TEM-1 | en_US |
| dc.subject.fr | protéine hybride | en_US |
| dc.subject.fr | insertion | en_US |
| dc.subject.fr | entérotoxine STa | en_US |
| dc.subject.fr | boucle V3 | en_US |
| dc.title | Creating hybrid proteins by insertion of exogenous peptides into permissive sites of a class A β-lactamase | en_US |
| dc.title.fr | Création de protéines hybrides par insertion de peptides exogènes dans des sites permissifs d'une β-lactamase de classe A | en_US |
| dc.type | Article scientifique | en_US |
| synhera.classification | Sciences du vivant>>Biochimie, biophysique & biologie moléculaire | en_US |
| synhera.institution | Autre | en_US |
| synhera.otherinstitution | Université de Liège, Centre d'Ingénierie des Protéines | en_US |
| synhera.otherinstitution | Université de Liège, Département des Maladies Infectieuse et Parasitaires | en_US |
| synhera.otherinstitution | Université Catholique de Louvain, Institut des Sciences de la Vie | en_US |
| synhera.otherinstitution | WIV-Pasteur Institute of Brussels, Mycobacterial Immunology | en_US |
| synhera.stakeholders.fund | ? | en_US |
| synhera.cost.total | ? | en_US |
| synhera.cost.apc | ? | en_US |
| synhera.cost.comp | ? | en_US |
| synhera.cost.acccomp | ? | en_US |
| dc.description.version | Oui | en_US |
| dc.rights.holder | Auteurs ; Federation of European Biochemical Societies (FEBS) (Utrecht) | en_US |
