Suppression of human cytochrome P450 aromatase activity by monoclonal and recombinant antibody fragments and identification of a stable antigenic complex.
Human cytochrome P450 aromatase (P450arom) is responsible for biosynthesis of estrogens from androgens. Monoclonal antibody MAb3-2C2 to P450arom specifically binds to a conformational epitope and suppresses the enzyme activity in a dose-dependent manner. The crystal structure of the Fab fragment of MAb3-2C2 has been used to engineer a recombinant single chain antibody fragment (scFv) and a homodimeric variable domain of the light chain (VL(2)). These recombinant antibody fragments have been expressed in Escherichia coli and purified. Here, we show that the recombinant scFv suppresses P450arom activity with an IC(50) value similar to that of natural MAb3-2C2 F(ab')(2). The recombinant VL(2) also exhibits dose-dependent suppression of the P450arom activity, but at a reduced level, demonstrating that the homodimer is unable to fully mimic the complementarity determining region (CDR) of a variable heavy chain (VH)-VL heterodimer. We prepare and purify a stable complex of P450arom with MAb3-2C2 F(ab')(2) and show that the complex migrates and precipitates as a single molecular assembly. Efforts to crystallize P450arom for structure-function studies have yielded small single crystals. Our results suggest that formation of stable complexes with fragments of the monoclonal antibody could provide an alternative method for crystallization of P450arom.
Lala
Puloma
P
Department of Structural Biology, Hauptman-Woodward Medical Research Institute, 73 High Street, Buffalo, NY 14203, USA.
Higashiyama
Tadayoshi
T
Erman
Mary
M
Griswold
Jennifer
J
Wagner
Traci
T
Osawa
Yoshio
Y
Ghosh
Debashis
D
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000941:Antigens / Q000276:immunology*; D001141:Aromatase / Q000276:immunology / Q000378:metabolism; D047072:Aromatase Inhibitors; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D000941:Antigens; D047072:Aromatase Inhibitors; D011994:Recombinant Proteins; D001141:Aromatase