Expression of Fab fragment of catalytic antibody 6D9 in an Escherichia coli in vitro coupled transcription/translation system

FEBS Letters

Nakano, Hideo²  Ookubo, Yuji¹  Jiang, XiuPing²  Yamane, Tsuneo²  Fujii, Ikuo¹ 

[1] Biomolecular Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan;Laboratory of Molecular Biotechnology, Graduate School of Biological and Agricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

关键词: Catalytic antibody;    Fab fragment;    Cell-free protein synthesis;    Antibody activity;    BSA;    bovine serum albumin;    CBB;    Coomassie brilliant blue;    ELISA;    enzyme-linked immunosorbent assay;    GSH;    glutathione reduced;    GSSG;    glutathione oxidized;    Hc;    heavy chain of Fab fragment;    HPLC;    high-performance liquid chromatography;    Lc;    light chain of Fab fragment;    PBS;    phosphate-buffered saline;    PCR;    polymerase chain reaction;    PDI;    protein disulfide-isomerase;    SDS–PAGE;    sodium dodecyl sulfate–polyacrylamide gel electrophoresis;   

DOI  :  10.1016/S0014-5793(02)02383-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown