Abstract
The heavy chain (Hc) and light chain (Lc) genes of the Fab fragment of a catalytic antibody 6D9 were simultaneously expressed in an Escherichia coli in vitro transcription/translation system without a reducing agent. The intermolecular disulfide bond between the Hc and Lc was found formed, suggesting a correct formation of the Fab fragment in the in vitro system. In enzyme-linked immunosorbent assay, the Fab fragment synthesized in vitro exhibited an antigen-binding activity. Addition of reduced glutathione, oxidized glutathione, protein disulfide-isomerase and molecular chaperones, GroEL and GroES, increased the solubility and the antigen-binding activity of the Fab fragment greatly. The in vitro synthesized Fab was purified by means of a hexa-histidine tag attached to the C-terminus of the Hc. Catalytic assay of the purified Fab fragment showed that the His-tagged Fab fragment synthesized in vitro had a catalytic activity comparable to that produced in vivo.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Antibodies, Catalytic / genetics*
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Antigens / metabolism
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Catalysis / drug effects
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Cell-Free System / chemistry
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Cell-Free System / metabolism
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Chaperonin 10 / pharmacology
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Chaperonin 60 / pharmacology
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Electrophoresis, Polyacrylamide Gel
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Enzyme-Linked Immunosorbent Assay
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Escherichia coli
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Glutathione / pharmacology
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Glutathione Disulfide / pharmacology
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Haptens / metabolism
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Immunoglobulin Fab Fragments / biosynthesis*
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Immunoglobulin Fab Fragments / chemistry*
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Immunoglobulin Fab Fragments / genetics
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Molecular Chaperones / pharmacology
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Oxidation-Reduction
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Protein Binding / drug effects
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Protein Biosynthesis / physiology
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Protein Disulfide-Isomerases / pharmacology
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Solubility / drug effects
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Transcription, Genetic / physiology
Substances
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Antibodies, Catalytic
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Antigens
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Chaperonin 10
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Chaperonin 60
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Haptens
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Immunoglobulin Fab Fragments
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Molecular Chaperones
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Protein Disulfide-Isomerases
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Glutathione
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Glutathione Disulfide