title: An FKBP12 binding assay based upon biotinylated FKBP12.
abstract: A binding assay was developed for measuring the affinity of FKBP12 ligands. A biotinylation signal sequence was fused to the 5' end of the human FKBP12 gene, and the fusion protein was expressed in Escherichia coli with biotin ligase. The fusion protein was immobilized in avidin-coated multiwell plates, and varying concentrations of test ligands were allowed to compete with [3H]FK506 for FKBP12 sites on the plate. The assay provided Kd values for FK520, 32-hydroxyethyl indolyl FK520, and 18-ene, 20-oxa FK520 that are in agreement with previously reported values. The assay provides a convenient and rapid method for the assessment of FKBP12 binding by small molecules.
authors.affiliation: Department of Pharmacological Sciences, Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, California 94545, USA. carreras@kosan.com
authors.forename: C W
authors.initials: CW
authors.lastname: Carreras
authors.forename: H
authors.initials: H
authors.lastname: Fu
authors.forename: D V
authors.initials: DV
authors.lastname: Santi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001667:Binding, Competitive / Q000502:physiology*; D019921:Biotinylation / Q000379:methods*; D019731:Carbon-Nitrogen Ligases / Q000235:genetics; D004800:Enzymes, Immobilized / Q000378:metabolism; D029968:Escherichia coli Proteins; D006801:Humans; D007700:Kinetics; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins; D016324:Sequence Tagged Sites; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000378:metabolism*; D022061:Tacrolimus Binding Protein 1A / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D022061:Tacrolimus Binding Protein 1A; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli; D016559:Tacrolimus