doi:10.1016/j.pep.2007.03.007
Crown copyright © 2007 Published by Elsevier Inc.
Expression of furin-linked Fab fragments against anthrax toxin in a single mammalian expression vector
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Christopher D. Reeda, Heidi Rasta, Wei-gang Hub, David Mahc, Les Nagatab and Saad A. Masria, 
, 
aCanadian Food Inspection Agency, Centre for Plant Health, 8801 East Saanich Road, Sidney, BC, Canada V8L 1H3
bChemical Biological Defence Section, Defence Research and Development Canada-Suffield, Box 4000, Station Main, Medicine Hat, Alta., Canada T1A 8K6
cCanada West Bioscience Inc., 113-339 50th Ave SE, Calgary, Alta., Canada T2G 2B3
Received 16 January 2007;
revised 10 March 2007.
Available online 21 March 2007.
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.
Keywords: Antibodies; Antibody expression Fab; Bacillus anthracis; Furin; Protective antigen
Fig. 1. Schematic diagram showing the construction of human Fab fragment containing the furin cleavage site.
Fig. 2. Western blot analysis of expressed anti-anthrax Fabs obtained from CHOK1 culture supernatant. The Fab fragments were purified on IMAC column, separated on 12.5% SDS–PAGE, and blotted in PVDF membranes. Lane 1: low-range molecular mass markers (Bio-Rad Cat. 161-0305). Lane 2: CHOK1 expressed light chain (before transfection with heavy chain construct) detected with anti-human Fab monoclonal antibody. Lane 3: CHOK1 expressed heavy and light chains (AAhuFab) detected with anti-human Fab (for light chain detection) and anti-His monoclonal antibodies (for heavy chain detection). Lane 4: CHOK1 Expressed AAhuFab-furin construct detected with anti-human Fab antibodies. Lane 5: CHOK1 Expressed AAhuFab-furin construct detected with anti-His monoclonal antibody.
Fig. 3. Western blot detection of rPA using purified Fab fragments. The Fab fragments were purified on IMAC column and tested on rPA which was separated on 12.5% SDS–PAGE, and blotted on PVDF membranes. The activity of the expressed Fab was detected by goat anti-human Fab antibodies. (a) Activity of the purified AAhuFab-furin. Lane 1: low-range molecular mass markers (Bio-Rad Cat. 161-0305). Lane 2: activity of the purified AAhuFab-furin fragment against negative control. Lane 3: activity of the purified AAhuFab-furin against rPA. (b) Activity of the purified AAhuFab obtained from the 2 plasmid expression system. Lane 1: low-range molecular mass markers (Bio-Rad). Lane 2: activity of the purified AAhuFab fragment against negative control. Lane 3: activity of the purified AAhuFab against rPA.
Table 1.
PCR Primers used to amplify and clone the Fab fragments, restriction endonuclease sites are listed in bold type
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