Cell-free production of functional antibody fragments

• Published in: Bioprocess and biosystems engineering Vol. 33; no. 1; pp. 127 - 132
• Main Authors: Oh, In-Seok, Lee, Ji-Chul, Lee, Myung-shin, Chung, Jun-ho, Kim, Dong-Myung
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 2010; Springer-Verlag; Springer; Springer Nature B.V
• Subjects: Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Biological and medical sciences; Biotechnology; Botulinum Toxins - immunology; Botulinum Toxins, Type A; Botulism; Cell-Free System; Chemical bonds; Chemical reactions; Chemistry; Chemistry and Materials Science; E coli; Environmental Engineering/Biotechnology; Escherichia coli; Escherichia coli - metabolism; Food Science; Fundamental and applied biological sciences. Psychology; Immunoglobulin Fab Fragments - biosynthesis; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - immunology; Industrial and Production Engineering; Industrial Chemistry/Chemical Engineering; Neurotoxins; Original Paper; Protein synthesis; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Redox potential; Toxicology; Botulinum neurotoxin; Cell-free protein synthesis system; Antigen-binding fragment (Fab); Disulfide bond; Antibody; Enzyme; Metalloendopeptidases; Fab-Fragment; Antigen; Peptidases; Bontoxilysin; Protein synthesis; Production; Hydrolases
• ISSN: 1615-7591; 1615-7605
• DOI: 10.1007/s00449-009-0372-3

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.