Expression of Fab fragment of catalytic antibody 6D9 in an Escherichia coli in vitro coupled transcription/translation system.
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.
Jiang
XiuPing
X
Laboratory of Molecular Biotechnology, Graduate School of Biological and Agricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Ookubo
Yuji
Y
Fujii
Ikuo
I
Nakano
Hideo
H
Yamane
Tsuneo
T
D017151:Antibodies, Catalytic / Q000235:genetics*; D000941:Antigens / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D018835:Chaperonin 10 / Q000494:pharmacology; D018834:Chaperonin 60 / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005978:Glutathione / Q000494:pharmacology; D019803:Glutathione Disulfide / Q000494:pharmacology; D006241:Haptens / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D018832:Molecular Chaperones / Q000494:pharmacology; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000187:drug effects; D014176:Protein Biosynthesis / Q000502:physiology; D019704:Protein Disulfide-Isomerases / Q000494:pharmacology; D012995:Solubility / Q000187:drug effects; D014158:Transcription, Genetic / Q000502:physiology
D017151:Antibodies, Catalytic; D000941:Antigens; D018835:Chaperonin 10; D018834:Chaperonin 60; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D018832:Molecular Chaperones; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D019803:Glutathione Disulfide