title: Construction of ribosome display library based on lipocalin scaffold and screening anticalins with specificity for estradiol.
abstract: 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 β-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 (IC(50)) for E(2) was 50 ng mL(-1) and the limit of detection (LOD:IC(10)) 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.
authors.affiliation: School of Chemical Engineering and Technology, Tian Jin University, Tianjin 300072, China.
authors.forename: Jianqing
authors.initials: J
authors.lastname: Liu
authors.forename: Baoan
authors.initials: B
authors.lastname: Ning
authors.forename: Ming
authors.initials: M
authors.lastname: Liu
authors.forename: Yanan
authors.initials: Y
authors.lastname: Sun
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Sun
authors.forename: Yihong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Xianjun
authors.initials: X
authors.lastname: Fan
authors.forename: Zhijiang
authors.initials: Z
authors.lastname: Zhou
authors.forename: Zhixian
authors.initials: Z
authors.lastname: Gao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D052244:Endocrine Disruptors / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D004958:Estradiol / Q000032:analysis*; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054834:Lipocalins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D052244:Endocrine Disruptors; D019476:Insect Proteins; D054834:Lipocalins; D019151:Peptide Library; D011994:Recombinant Proteins; C053849:bilin-binding protein, insect; D004958:Estradiol