Cloning, sequencing, expression and characterization of three anti-estradiol-17beta Fab fragments.
In order provide data for a basic understanding of the mechanisms of antibody specificity and for the design of antibodies with desired properties, we have sequence-analysed three high affinity anti-estradiol-17beta monoclonal antibodies. All three monoclonal antibodies to estradiol-17beta had been raised by conjugation of the 6-carboxymethyloxime derivative to protein carrier. The genes encoding heavy (Fd) and light (L) chains of these three antibodies were cloned and sequenced. The sequenced antibody chains were found to be from 46.0 to 89.7% sequence identical to a monoclonal antibody (DB3) binding a related steroid, progesterone. The Fd and L chains were paired with all possible Fd-L combinations and the corresponding proteins were expressed in Escherichia coli and characterized for their binding (immunoreactivity) to estradiol-17beta. Under the lac promoter and using the pelB signal sequences the production levels of the soluble (total) heavy and light chain Fab fragment combinations in periplasm and in supernatant varied from 115 to 2207 microg/l, while the immunoreactivity percentages (IR%) varied from < 1 to 45%. The production levels and IR% were dependent on the first constant domain subclasses of the heavy chain as well as the Fd-L chain combination expressed.
Pajunen
M
M
Department of Biotechnology, University of Turku, Finland. maria.pajunen@btk.utu.fi
Saviranta
P
P
Jauria
P
P
Karp
M
M
Pettersson
K
K
Mäntsälä
P
P
Lovgren
T
T
D000911:Antibodies, Monoclonal / Q000235:genetics; D000918:Antibody Specificity / Q000235:genetics*; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D004958:Estradiol / Q000276:immunology*; D017578:Immunoglobulin Class Switching; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D007127:Immunoglobulin Constant Regions; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D004958:Estradiol