Cell-free production of functional antibody fragments.
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.
Oh
In-Seok
IS
Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea.
Lee
Ji-Chul
JC
Lee
Myung-shin
MS
Chung
Jun-ho
JH
Kim
Dong-Myung
DM
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
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A