title: Efficient extracellular production of hybrid E. coli heat-labile enterotoxin B subunits in a marine Vibrio.
abstract: Escherichia coli heat-labile enterotoxin B subunit (EtxB) has been proposed as a potential protein carrier for the delivery of heterologous peptides to target cells, particularly for the oral delivery of epitopes to the mucosal immune system. In this study, two extensions to the C-terminus of EtxB were genetically engineered that correspond to a well-characterized neutralising epitope of glycoprotein D from herpes simplex virus (EtxB-gD) and to the C-terminal nine amino acids from the 38 kDa subunit of HSV-encoded ribonucleotide reductase (EtxB-R2). Here we describe the extracellular secretion of the two hybrid EtxBs from a marine Vibrio harbouring a broad-host range inducible expression vector containing the hybrid genes. Large amounts of intact fusion proteins (15-20 mg per liter of culture) were secreted into the medium upon induction. These hybrid proteins maintained the receptor-binding activity of the native toxin as well as being cross-reactive with anti-EtxB and anti-heterologous peptide monoclonal antibodies.
authors.affiliation: Institute of Microbiology, University of Padua, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Marcello
authors.forename: A
authors.initials: A
authors.lastname: Loregian
authors.forename: G
authors.initials: G
authors.lastname: Palù
authors.forename: T R
authors.initials: TR
authors.lastname: Hirst
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism; D004768:Enterotoxins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005822:Genetic Vectors; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D014733:Vibrio / Q000235:genetics / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C027538:heat-labile enterotoxin, E coli; D010450:Endopeptidases