title: Cell-free production of functional antibody fragments.
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 microg of functional Fab was obtained after purification from 1 mL reaction mixture.
authors.affiliation: Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea.
authors.forename: In-Seok
authors.initials: IS
authors.lastname: Oh
authors.forename: Ji-Chul
authors.initials: JC
authors.lastname: Lee
authors.forename: Myung-shin
authors.initials: MS
authors.lastname: Lee
authors.forename: Jun-ho
authors.initials: JH
authors.lastname: Chung
authors.forename: Dong-Myung
authors.initials: DM
authors.lastname: Kim
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D001905:Botulinum Toxins / Q000276:immunology; D019274:Botulinum Toxins, Type A; D002474:Cell-Free System; D004926:Escherichia coli / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A