Cloning, sequencing, expression and characterization of three anti-estradiol-17β Fab fragments

摘要

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-17β monoclonal antibodies. All three monoclonal antibodies to estradiol-17β 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-17β. 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 μg/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.