Production of stable anti-digoxin Fv in Escherichia coli.
We have created a bacterial expression-export system and have used it to express (14 mg l-1) the variable region fragment (Fv) of an anti-digoxin antibody (26-10) in Escherichia coli. The expression-export plasmid contains a T7 promoter and the E. coli signal sequences ompA [Movva et al., J. biol. Chem. 255, 27-29 (1980)] and phoA [Inouye et al., J. Bacteriol. 149, 434-439 (1982)] fused to heavy chain (VH) and light chain (VL) variable region sequences to generate an artificial cistron. The 26-10 Fv protein made using this system was soluble, unlike many other expression systems which produce insoluble proteins in the form of inclusion bodies. The 26-10 VH and VL proteins were cleaved at their mature N-termini and exported into the bacterial periplasm where they could be easily extracted and affinity purified on ouabain-Sepharose. 26-10 Fv bound to digoxin with similar affinity and specificity as the whole 26-10 antibody (Ka for Fv, 1.3 x 10(9) M-1, Ka for IgG, 7 x 10(9) M-1). 26-10 Fv appears to be remarkably stable in comparison with other Fv fragments. The half-life for chain dissociation of 26-10 Fv was 48 hr compared to the reported 1.5 hr half-life of McPC603 Fv. We present the proton NMR spectra of the 26-10 Fv as preliminary evidence that this expression-export system can be used to facilitate the analysis of the solution structure of 26-10 Fv by NMR.
Anthony
J
J
Bristol-Myers Squibb Pharmaceutical Research Institute, Department of Molecular Biology of Macromolecules, Princeton, NJ 08543.
Near
R
R
Wong
S L
SL
Iida
E
E
Ernst
E
E
Wittekind
M
M
Haber
E
E
Ng
S C
SC
D000595:Amino Acid Sequence; D000915:Antibody Affinity; D000918:Antibody Specificity; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004077:Digoxin / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000276:immunology*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D007074:Immunoglobulin G / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000502:physiology; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D004077:Digoxin