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.
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.
Selisko
Barbara
B
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.
Cosío
Gabriela
G
García
Consuelo
C
Becerril
Baltazar
B
Possani
Lourival D
LD
Horjales
Eduardo
E
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*
D000911:Antibodies, Monoclonal; D000997:Antivenins; C035413:Centruroides toxin; D007140:Immunoglobulin Fab Fragments; D009498:Neurotoxins; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms