ABSTRACT
Recombinant immunoconjugates of marker enzymes with antigens or antibodies present considerably more advantages than those obtained by conventional methods of chemical synthesis; i.e. they are homogeneous, have a strictly determined stoichiometry, and retain the functional activity of both a marker protein and an antigen/antibody. Based on the pPICZαB shuttle vector, we first managed to obtain a recombinant conjugate of key marker enzyme horseradish peroxidase (HRP) withFabfragments of antibodies against atrazine. The resulting genetic construction allows us to switch to any other antibody sequence, via the simple re-cloning of variable parts and an additional reporter enzyme. Conjugates were successfully produced in thePichia pastorismethylotrophic yeast expression system. The target activity of the conjugates (both enzymatic and antigen-binding) has been demonstrated by ELISA method.
ABSTRACT
Models of three-dimensional structures of Fv domains of three antibodies specific to the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) have been derived from the sequence data by comparative modeling. The same binding site cavities were observed in all cases. The most important residue in antigen binding is tyrosine, which serves as a wall of cavity and putatively forms pi-stacking interaction with aromatic moiety of the ligand. Another cavity wall is formed by hydrophobic residues. At the entrance of cavity a glutamate residue is located in 2 of 3 structures. Docking of 2,4-D and its analogs on the models was performed. On the basis of docking results an experimental cross-reactivity data were qualitatively explained. Using results of the modeling, mutation of glutamate to serine or lysine was suggested to eliminate electrostatic repulsion between antibody and ligand and to improve 2,4-D binding efficiency. Target mutations in the antibody binding site were checked on the model.