Escherichia coli Secretion of an Active Chimeric Antibody Fragment

Abstract

A chimeric mouse-human Fab protein that binds specifically to the human carcinoma cell line C3347 has been expressed and secreted from Escherichia coli. This molecule, which contains functionally assembled kappa and Fd proteins, binds as effectively to sites on the surface of C3347 cells as Fab fragments prepared proteolytically from whole chimeric or mouse antibody. The production in Escherichia coli of foreign heterodimeric protein reagents, such as Fab, should prove useful in the management of human disease.

Broad-Host-Range Plasmid pJB658 Can Be Used for Industrial-Level Production of a Secreted Host-Toxic Single-Chain Antibody Fragment in Escherichia coli

“…From these cultures, cells were used to inoculate 0.75 liter of prewarmed main culture medium in the fermentor (3- High-cell-density cultivation. All fermentations were performed at 30°C, and the pH was maintained at 6.8 by addition of 12.5% (vol/vol) NH 3 . Antifoam (Adecanol LG-109; Asahi Denka Kogyo, Japan) was added from the start (40 l/liter) and thereafter when needed.…”

“…Although expression of native scFv proteins without disulfide bridge formation has been reported (9), cytoplasmic production in bacteria typically results in aggregation of scFv polypeptides into insoluble inclusion bodies (14). Therefore, in E. coli it is usually desirable to express scFvs as fusion proteins targeted for translocation to the oxidative periplasm to obtain functional products (3,26). Various vector systems for recombinant scFv expression in E. coli have been reported (11,15,19), but the experiments were typically performed in shake-flask cultures with product yields of 10 to 30 mg/liter.…”

Leaderless polypeptides efficiently extracted from whole cells by osmotic shock

“…In addition to the well-documented translocation of proteins from cytoplasm to periplasm, selective protein localization to a variety of subcellular structures and organelles in E. coli has previously been documented (2,6,7,45,54,60); this has been observed even for proteins that are not native to cells (30,35,56). The mechanisms which mediate these localization events in bacterial cells remain to be fully elucidated.…”

“…During translocation, this leader is generally cleaved from the mature protein (53, 67). The absence or inactivation of this leader by mutation results in a protein that cannot be efficiently translocated out of the cytoplasm by the cell's general secretory apparatus unless compensatory mutations are simultaneously introduced into one or more of the cell's secretory apparatus components (10,19,23,47).In addition to the well-documented translocation of proteins from cytoplasm to periplasm, selective protein localization to a variety of subcellular structures and organelles in E. coli has previously been documented (2,6,7,45,54,60); this has been observed even for proteins that are not native to cells (30,35,56). The mechanisms which mediate these localization events in bacterial cells remain to be fully elucidated.…”

Uniform labeling of a recombinant antibody Fv‐fragment with 15N and 13C for heteronuclear NMR spectroscopy

“…In complex systems, resonance overlap in homonuclear proton spectra can be resolved by the use of heteronuclear experiments performed with "Nand 13C-enriched samples, relying on the greater chemical shift dispersion of the heteronucleus [ 11. Concurrently, progress in protein engineering has enabled the production of functional recombinant antibody-subunits in E. coli [2,3] and eukaryotic cells [4]. The smallest antibody fragment which still retains the natural antigen binding site is the so-called Fv-fragment.…”