Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen.
A 1460-bp DNA encoding the two chains of the antigen-binding fragment (Fab) portion of a monoclonal antibody have been chemically synthesized and expressed in Escherichia coli. The antibody, Se155-4, is specific for a Salmonella serogroup B O-antigen and its crystal structure is under investigation. The genes were synthesized according to a strategy that allows for easy manipulation in genetic engineering studies of the Fab-binding site. Each gene is preceded by the ompA secretory signal and a ribosome-binding site, and has been expressed from the two-cistron DNA under the control of the lac promoter. Active Fab of 50 kDa with an inter-chain disulfide bond has been isolated from the periplasm of E. coli in a one-step affinity purification in high yield (2 micrograms/ml of cells). The bacterially produced Fab is as active as purified mouse Fab in antigen-binding and competitive immunoassays. This is the first example of a completely synthetic Fab gene and provides an ideal system to probe the nature of antigen binding by anti-carbohydrate antibodies.
Anand
N N
NN
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Dubuc
G
G
Phipps
J
J
MacKenzie
C R
CR
Sadowska
J
J
Young
N M
NM
Bundle
D R
DR
Narang
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005803:Genes, Immunoglobulin; D005813:Genes, Synthetic; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D019081:O Antigens; D015345:Oligonucleotide Probes; D010957:Plasmids; D015183:Restriction Mapping; D012475:Salmonella / Q000145:classification / Q000276:immunology*; D012689:Sequence Homology, Nucleic Acid; D012703:Serotyping
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D007140:Immunoglobulin Fab Fragments; D019081:O Antigens; D015345:Oligonucleotide Probes