Construction of ribosome display library based on lipocalin scaffold and screening anticalins with specificity for estradiol

A new anticalin against estradiol (E-2), a kind of endocrine disruptor, was obtained in the present study to detect E-2 levels. A member of the lipocalin family from Pieris brassicae called bilin-binding protein (BBP) was employed for the preparation of a random library to specifically complex E-2. Sixteen amino acid residues at the center of the binding site, which were formed by four loops on top of an eight-stranded beta-barrel, were subjected to targeted random mutagenesis. Estradiol-binding BBP variants so-called 'anticalins', which exhibit binding activity for compounds, such as E-2, were selected from the resulting library by combining both ribosome display and screening techniques. Four variants of complex E-2 with high affinity were identified. These variants exhibited dissociation constants (KDs) as low as 54.265 nM. ELISA showed that ribosome displayed anticalin (E-2-A) specifically bound E-2. The 50% inhibition concentration (IC50) for E-2 was 50 ng mL(-1) and the limit of detection (LOD:IC10) was 0.071 ng mL(-1). The experimental results suggest that E-2-A can be used as a potential anticalin to detect E-2 in animals.