Cell-free production of functional antibody fragments |
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Authors: | In-Seok Oh Ji-Chul Lee Myung-shin Lee Jun-ho Chung Dong-Myung Kim |
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Institution: | (1) Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea;(2) Abxign, Inc., Samsung Cancer Research Building, Seoul, Korea;(3) Department of Microbiology and Immunology, Eulji University School of Medicine, Taejon, Korea;(4) Department of Biochemistry and Molecular Biology, Cancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea;(5) Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Taejon, 305-764, Korea; |
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Abstract: | Botulinum neurotoxin serotype B (BoNT/B)-specific Fab was expressed in a cell-free protein synthesis system derived from an
E. coli extract. The cell-free synthesized antibody fragment was found to be effective in neutralizing the toxicity of BoNT/B in
animal studies. Expression of functional Fab required an appropriately controlled and stably maintained redox potential. Under
an optimized redox condition, the cell extract, whose disulfide reducing activity had been exhausted, could generate bio-functional
Fab molecules. Use of a cell extract enriched with molecular chaperones (GroEL/ES) and disulfide bond isomerases were effective
in obtaining larger quantities of functional Fab. Under the optimized reaction conditions, approximately 30 μg of functional
Fab was obtained after purification from 1 mL reaction mixture. |
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