title: Creating hybrid proteins by insertion of exogenous peptides into permissive sites of a class A beta-lactamase.
abstract: 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 beta-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 beta-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.
authors.affiliation: Biological Macromolecules and Laboratory of Enzymology, Centre d'Ingénierie des Protéines, University of Liège, Belgium.
authors.forename: Nadia
authors.initials: N
authors.lastname: Ruth
authors.forename: Birgit
authors.initials: B
authors.lastname: Quinting
authors.forename: Jacques
authors.initials: J
authors.lastname: Mainil
authors.forename: Bernard
authors.initials: B
authors.lastname: Hallet
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Frère
authors.forename: Kris
authors.initials: K
authors.lastname: Huygen
authors.forename: Moreno
authors.initials: M
authors.lastname: Galleni
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D000917:Antibody Formation / Q000187:drug effects; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology*; D004768:Enterotoxins / Q000235:genetics / Q000276:immunology*; D029968:Escherichia coli Proteins; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000276:immunology; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*; D012995:Solubility; D014612:Vaccines / Q000138:chemical synthesis*; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics*
chemical_list: D000907:Antibodies, Bacterial; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D015699:HIV Envelope Protein gp120; D011993:Recombinant Fusion Proteins; D014612:Vaccines; C034151:heat stable toxin (E coli); D001618:beta-Lactamases; C034413:beta-lactamase TEM-1