title: Expression of furin-linked Fab fragments against anthrax toxin in a single mammalian expression vector.
abstract: Human anti-recombinant protective antigen (rPA) Fab genes were previously cloned from single B cells of a donor immunized with anthrax vaccine using fluorescence activated cell sorting with fluorescein labeled rPA and single-cell PCR. The light and heavy chains were sub-cloned individually into mammalian expression vectors pSecTag2B or pEXPR44, respectively, and expressed in the same CHOK1 cells. Alternatively, the same heavy and light chains were linked together, using PCR, with an in-frame sequence coding for a furin cleavage site. This construct was cloned into pSecTag2B and expressed in CHOK1 cells. Once expressed, the individual chains combined in vivo to form a Fab fragment which was purified as a single protein when either method was utilized. The human Fab antibodies produced by this technique were functional when tested in Western blots using the recombinant PA antigen as the target.
authors.affiliation: Canadian Food Inspection Agency, Centre for Plant Health, 8801 East Saanich Road, Sidney, BC, Canada V8L 1H3.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Reed
authors.forename: Heidi
authors.initials: H
authors.lastname: Rast
authors.forename: Wei-Gang
authors.initials: WG
authors.lastname: Hu
authors.forename: David
authors.initials: D
authors.lastname: Mah
authors.forename: Les
authors.initials: L
authors.lastname: Nagata
authors.forename: Saad A
authors.initials: SA
authors.lastname: Masri
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D045683:Furin / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis*; D014162:Transfection
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D007140:Immunoglobulin Fab Fragments; C030325:anthrax toxin; D045683:Furin