Engineering of Escherichia coli beta-galactosidase for solvent display of a functional scFv antibody fragment.
Protein engineering allows the generation of hybrid polypeptides with functional domains from different origins and therefore exhibiting new biological properties. We have explored several permissive sites in Escherichia coli beta-galactosidase to generate functional hybrid enzymes displaying a mouse scFv antibody fragment. When this segment was placed at the amino-terminus of the enzyme, the whole fusion protein was stable, maintained its specific activity and interacted specifically with the target antigen, a main antigenic determinant of foot-and-mouth disease virus. In addition, the antigen-targeted enzyme was enzymatically active when bound to the antigen and therefore useful as a reagent in single-step immunoassays. These results prove the flexibility of E. coli beta-galactosidase as a carrier for large-sized functional domains with binding properties and prompt the further exploration of the biotechnological applicability of the scFv enzyme targeting principle for diagnosis or other biomedical applications involving antigen tagging.
Alcalá
Pilar
P
Institut de Biotecnologia i de Biomedicina and Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Barcelona, Spain.
Ferrer-Miralles
Neus
N
Villaverde
Antonio
A
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000235:genetics / Q000378:metabolism; D000918:Antibody Specificity; D000956:Antigens, Viral; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D029544:Foot-and-Mouth Disease Virus / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012997:Solvents; D001616:beta-Galactosidase / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism
D000914:Antibodies, Viral; D000956:Antigens, Viral; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D012997:Solvents; D001616:beta-Galactosidase