title: Bacterial expression, purification and functional characterization of a recombinant chimeric Fab derived from murine mAb BCF2 that neutralizes the venom of the scorpion Centruroides noxius hoffmann.
abstract: The murine monoclonal antibody BCF2 is able to neutralize the venom of the scorpion Centruroides noxius Hoffmann. A chimeric Fab of BCF2 (chFab-BCF2) comprising the variable regions of murine BCF2 and human constant regions was assembled. chFab-BCF2 was expressed as a soluble and functional protein in the periplasmic space of Escherichia coli. An expression yield of 1 mg/l was reached by combination of late-log-phase induction, rich culture medium, low expression temperature and addition of sucrose (0.3 M) to the culture medium. The addition of sucrose induced secretion of 60% of the protein into the medium. After expression for 23 h, a novel process was used to release the remaining periplasmic protein in situ consisting in the addition of lysozyme and sucrose up to 0.6 M (20%) directly to the culture medium. chFab-BCF2 was recovered by ammonium sulfate precipitation and purified in a single step by affinity chromatography using anti-human anti-F(ab')(2) IgG coupled to Sepharose-proteinG. Pure chFab-BCF2 maintained a similar nanomolar affinity as BCF2 to its cognate antigen, the Na(+)-channel-affecting toxin Cn2. Recombinant chFab-BCF2 was able to neutralize Cn2 in vivo even at a molar ratio of 1:1, as well as the whole venom of C. noxius. Thus, it is a promising candidate to be used as a specific and efficient recombinant antidote against scorpion stings.
authors.affiliation: Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology/National Autonomous University of Mexico (UNAM), Av. Universidad 2001, Apartado Postal 510-3, Cuernavaca, Morelos 62210, Mexico.
authors.forename: Barbara
authors.initials: B
authors.lastname: Selisko
authors.forename: Gabriela
authors.initials: G
authors.lastname: Cosío
authors.forename: Consuelo
authors.initials: C
authors.lastname: García
authors.forename: Baltazar
authors.initials: B
authors.lastname: Becerril
authors.forename: Lourival D
authors.initials: LD
authors.lastname: Possani
authors.forename: Eduardo
authors.initials: E
authors.lastname: Horjales
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D000997:Antivenins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D051379:Mice; D009498:Neurotoxins / Q000276:immunology; D009500:Neutralization Tests; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012604:Scorpion Venoms / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000997:Antivenins; C035413:Centruroides toxin; D007140:Immunoglobulin Fab Fragments; D009498:Neurotoxins; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms