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

A new anticalin against estradiol (E2), a kind of endocrine disruptor, was obtained in the present study to detect E2 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 E2. Sixteen amino acid residues at the center of the binding site, which were formed by four loops on top of an eight-stranded β-barrel, were subjected to targeted random mutagenesis. Estradiol-binding BBP variants so-called ‘anticalins’, which exhibit binding activity for compounds, such as E2, were selected from the resulting library by combining both ribosome display and screening techniques. Four variants of complex E2 with high affinity were identified. These variants exhibited dissociation constants (KDs) as low as 54.265 nM. ELISA showed that ribosome displayed anticalin (E2–A) specifically bound E2. The 50% inhibition concentration (IC50) for E2 was 50 ng mL−1 and the limit of detection (LOD:IC10) was 0.071 ng mL−1. The experimental results suggest that E2–A can be used as a potential anticalin to detect E2 in animals.