Improved bivalent miniantibodies, with identical avidity as whole antibodies, produced by high cell density fermentation of Escherichia coli.
The combination of single-chain Fv-fragments (scFv) with a C-terminal, flexible linking region followed by a designed or natural dimerization domain provides a versatile system for targeted association of functional fragments in the periplasmic space of Escherichia coli. For homodimerization in vivo, two scFv fragments with a C-terminal hinge followed by a helix-turn-helix motif form "miniantibodies" with significantly higher avidity than in the case of leucine zipper containing constructs. The favorable design probably results in an antiparallel four-helix bundle and brings the homodimer to the same avidity as the whole IgA antibody, from which the binding site was taken. The molecular weight of the bivalent miniantibody is almost the same as that of a monovalent Fab fragment. We report here a high-cell density fermentation of E. coli producing these miniantibodies and a work-up procedure suitable for large scale production. Without any need of subsequent chemical coupling in vitro, approximately 200 mg/l of functional dimeric miniantibodies can be directly obtained from the E. coli culture.
Pack
P
P
Max-Planck-Institut für Biochemie, Protein Engineering Group, Martinsried, Fed. Rep. Germany.
Kujau
M
M
Schroeckh
V
V
Knüpfer
U
U
Wenderoth
R
R
Riesenberg
D
D
Plückthun
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D003001:Cloning, Molecular / Q000379:methods*; D015195:Drug Design; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000254:growth & development*; D005285:Fermentation; D005821:Genetic Techniques; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000737:chemistry / Q000145:classification; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis* / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000737:chemistry; D046911:Macromolecular Substances; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D007074:Immunoglobulin G; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D046911:Macromolecular Substances; D011994:Recombinant Proteins