Modulating the binding properties of an anti-17beta-estradiol antibody by systematic mutation combinations.
The anti-17beta-estradiol antibody 57-2 has been a subject for several protein engineering studies that have produced a number of mutants with improved binding properties. Here, we generated a set of 16 antibody 57-2 variants by systematically combining mutations previously identified from phage display-derived improved antibody mutants. These mutations included three point mutations in the variable domain of the light-chain and a heavy-chain variant containing a four-residue random insertion in complementarity determining region CDR-H2. The antibody variants were expressed as Fab fragments, and they were characterized for affinity toward estradiol, for cross-reactivity toward three related steroids, and for dissociation rate of the Fab/estradiol complex by using time-resolved fluorescence based immunoassays. The double-mutant cycle method was used to address the cooperativity effects between the mutations. The experimental data were correlated with structural information by using molecular modeling and visual analysis of the previously solved antibody 57-2 crystal structures. These analyses provided information about the steroid-binding mode of the antibody, the potential mechanisms of individual mutations, and their mutual interactions. Furthermore, several combinatorial mutants with improved affinity and specificity were obtained. The capacity of one of these mutants to detect estradiol concentrations at a clinically relevant range was proved by establishing a time-resolved fluorescence based immunoassay.
Department of Biotechnology, University of Turku, 20520 Turku, Finland. urpo.lamminmaki@utu.fi
Urpo
U
Lamminmäki
Annette
A
Westerlund-Karlsson
Maria
M
Toivola
Petri
P
Saviranta
D000906:Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000918:Antibody Specificity; D001666:Binding Sites, Antibody / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D004958:Estradiol / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D011487:Protein Conformation; D013256:Steroids / Q000737:chemistry
D000906:Antibodies; D007135:Immunoglobulin Variable Region; D013256:Steroids; D004958:Estradiol